Model studies in bio-organic processes : sodium transport
across biological membranes : an experimental study :
quantum chemical calculations on the stereochemistry of
coenzyme B12 dependent carbon-skeleton rearrangements
Citation for published version (APA):Merkelbach, I. I. (1985). Model studies in bio-organic processes : sodium transport across biological membranes : an experimental study : quantum chemical calculations on the stereochemistry of coenzyme B12 dependent carbon-skeleton rearrangements. Technische Hogeschool Eindhoven. https://doi.org/10.6100/IR179116
DOI:
10.6100/IR179116
Document status and date: Published: 01/01/1985
Document Version:
Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers)
Please check the document version of this publication:
• A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website.
• The final author version and the galley proof are versions of the publication after peer review.
• The final published version features the final layout of the paper including the volume, issue and page numbers.
Link to publication
General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain
• You may freely distribute the URL identifying the publication in the public portal.
If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement:
www.tue.nl/taverne Take down policy
If you believe that this document breaches copyright please contact us at: openaccess@tue.nl
MODEL STUDIES IN BIO-ORGANIC PROCESSES.
Sodiun1 ion transport across biologica! membranes.
An
experimental study.
Quanturn chemical calculations on the stereochemistry of
coenzyn1e B
12
dependent carbon-skeleton rearrangements.
MODEL STlTDIES IN BIO-ORGANIC PROCESSES.
Sodium ion transport across biological membranes.
An experimental study.
Quanturn chemical calculations on the stereochemistry of
coenzyme B
12
dependent carbon-skeleton rearrangements.
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECHNISCHE HOGESCHOOL EINDHOVEN, OP GEZAG VAN DE RECTOR MAGNIFICUS, PROF. DR. S.T.M. ACKERMANS, VOOR EEN COMMISSIE AJ,NGEWEZEN DOOR HET COLLEGE VAN DEKANEN IN HET OPENBAAR TE VERDEDIGEN OP VRIJDAG 10 MEI 1985 TE 16.00 UUR
DOOR
INGRID IRENE MERKELSACH
DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE PROMOTOREN
PROF. DR. H.M. BOCK EN
COlft'ENTS
Sodium ion transport across biologica! membranes. An experimental study. I . Introduction. I . 1 • I . 2. I . 3. I . 4. Membrane properties.
Membrane function. Scope of the first part of this thesis.
Developments in organophosphorus chemistry. Transfer of conformational changes in a phosphate group to the hydrophobic part of organic molecules.
References and notes.
I I . Sodium ion transport across biologica! membranes. 9 11 1 4 18 22 !1.1. Theory. 25
!1.1.1. Cluster formation in biological membranes 25 induced via a phospholipid P(V) TBP
intermediate.
!!.1.2. Activatien of membrane proteins in phospholipid clusters. II.2. Experiments. II.2.1. Introduction. II.2.2. Results. 11.2.3. Discussion. II.2.4. Conclusion. II.2.5. Experimental.
References and notes.
by uptake 33 37 37 40 43 50 50 53
Quantum chemical calculations on the stereochemistry of coenzyme 812-dependent carbon-skeleton rearrangements. III. ~e formation of a carbanionic intermediate
in the carbon-skeleton rearrangement step. III.l Structure and function of coenzyme B12• III.2. Mechanism of action of the carbon-skeleton
rearrangements.
III.3. The stereochemistry of the carbon-skeleton rearrangements as 'test' for the carbanionic machanism. Scope of the second part of this thesis.
III.4. The nature of the hydrogen transferred temporarily to coenzyme B12 during the carbon-skeleton rearrangements.
References and notes.
IV. Quantum chemical calculations.
IV. 1. IV. 2. IV. 3. IV. 4. IV. 5. Introduction.
The choice of the calculational method. Results.
Discussion. Conclusion.
References and notes1
SUIIIlllary Samenvatting Curriculum Vitae Dankwoord 59 62 65 67 69 73 74 76 85 87 88 89. 91 93 94
Voor het slagen van het kwaad is niets anders nodig dan dat de goede mensen niets doen.
Sodium ion transport across biologica! membranes. An experimental study.
I. Introduction.
I.l. Membrane properties.
Biologica! membranes play a crucial role in almost all cellular phenomena, yet our understanding of the molecular organization of membranes still can be called far from exhaustive. While the composition of membranes varies with their source, they generally contain approximately 40% of their dry weight as lipid and 60% as protein1. Usually carbohydrate is present to the extent of 1-10% of the total dry weight. In addition to these components, membranes contain some 20% of their total weight as water, which is tightly bound and essential to the maintenance of their
I.l. The fluid mosaic mode of Singerand Niaolson. Integral proteins (crossing the lipid bilayer), pheripheral proteins (bound to the exterior the bilayer) and proteins embedded in the matrix are bound to a functional complex or dissolved individually in the membrane bilayer.
structure. These components are organized according to the fluid mosaic model of Singer and Nicolson2 (see Figure I.1.). The lipids span a discontinuous bilayer, with their hydrophobic tails pointing towards the interior of the membranes and their hydrophilic head groups in contact with the water phase outside the membrane. In this bilayer
integral proteins are embedded, occasionally crossing the total lipid bilayer matrix, while pheripheral proteins are bound exterior to the bilayer. Dependent on e.g. temperature and water content3, the more or less extended hydracarbon ebains of the lipide tilt away from the perpendicular to the plane of the membrane, thus changing the ratio of the cross sectional area~ of head group and chain region4. In this way a modification in density of the membrane can be reached, comparable to the melting phenomena of classica! chemica! compounds, e.g. from a fluid liquid-crystalline to a solid gel-like phase. This melting can, even for pure lipids, not be described as a thermodynamic first-order phase transition, since the transition is certainly not discontinuous, as
shown, for example, by measurements of volume changes5. One can imagine a gel-like phase in which an appreciable lateral diffusion of the lipid molecules exists, while on increasing temperature this lateral diffusion will be accelerated
throughout a phase-transition region to the fluid liquid-crystalline phase. The width of this phase-transition region will be increased when mixing different lipids with each other, with proteins or with other membrane constituents5. In natura! membranes local gel-like domains6 exist over a large temperature range in liquid-crystalline matrices and vice-versa. These domains, often called clusters, develop in a continuing process of ordering and successive relaxation to a disordered state. They consist of mainly lipide, mainly
proteins or a mixture of both, but always can be described as a region of different density compared to the surrounding matrix. Diffusion over longer distances sametimes will be
opposed in biologica! membranes by a cytoskeletal system locallzing essential proteins in a well-defined region and by
a number of multivalent ligands that can induce aggregation of proteins into clusters, patches or caps? in an alternative way as mentioned above. Here proteins reside in a defined region near to each other, but are not necessarily located in one and the same cluster in the sense of same degree of order or same density. When the word cluster is used throughout this thesis, a small region of one aggregational state in a matrix of another will be ment, the first description of clusters given above.
I . 2. Membrane function. Scope of the first part of this
thesis.
There is a lot of controversy concerning the role of lipids in a membrane. Some authors state that lipids are the
insulating constituents of the membrane, separating different cellular compartments8. They form the structural support of membrane proteins, thus maintaining a constant spatial relationship between them. In this view ion transport across
~tf!!il\
~~
plasma membrane Golgi membrane rough endoplasmie reticulum
nuclear membt'lli'le outer mitochondria! membrane inner mitochondria! membrane
I.2. Lipid aomposition by weight of different subcellu~ membranes rat liver; PL phospholipid; C cholesterol; G glycolipid; unlabel-led= mono-> and triacylglycerols, free fatty acids and stearyl esters.
plasma membtane Golgi membtane rough endoplasmie reticulum
w
PSnuclear membtane outer mitochondria! membrane inner mitochondria! membrane
Figure I.3. Phospholipid aomposition by weight of different subaelluLar membranes of rat Ziver; PC = phosphatidyZahoZine; PE = phosphatidyZethanoZ-amine; PI = phosphatidylinositoZ; PS = phosphatidylserine; S sphingo-myeZin.
membranes is a property of integral proteins, that are influenced by external factors like metal-ions, protons, potential field etc. If one conaiders however, that lipid composition of animal membranes vary both with their tissue souree and intracellular location9 (see Figure I.2 and I.3), the question is raised whether the lipids could play a role themselves. So it has been suggested that the level of free fatty acids, that varies with the functional state of the membrane, may be involved in changes in membrane permeabil-ity10. Until now, little attention has been paid to the varying amounts of phospholipids in the membranes, in
particular to the·transition of the four co-ordinated to the five co-ordinated state of the phosphate group, bound on its crucial position between the polar headgroup and hydrophobic hydracarbon region, in relation to the phenomenon of ion transport. Recent studies on a number of model compounds for biologica! reactives11,12 suggest a general principle in passing biologica! information from ionic polar regions to hydrocarbon zones of natura! molecules via a five co-ordi-nated (P(V)) trigonal bipyramidal (TBP} intermediate (see
Cbapter !.3. and !.4.). A classica! four co-ordinate (P(IV)) tetragonal intermediate, possesses four ligands tbat are arranged spberically around tbe pbospborus atom, i.e. tbe P-L bond lengtbs and tbe L-P-L bond angles are equal. Wben a five co-ordinated TBP intermediate is formed{ a structural
inequivalence between two types of ligands (tbe equatorial and the axial ones) is introduced, since five ligands can not be spherically arranged around one atom (see Figure 1.4.).
T
TBP
I. 4. The four co-or'dir.ated tetY>aeder (T J and the co-ordinated
(TBP) of phosphorus.
The axial ligands {an incoming group e.g. water and the group through wbich tbe pbosphate is bound to tbe main chain of the biomolecule) are more electron withdrawing groups (see
Chapter !.3.) tban the equatorial ones, thus inducing an electron flux into tbe axis of the TBP. 1f the group in the axis consists of tbe 0-C-C-0-sequences often encountered in biomolecules, this extra negative charge on the axial oxygen
(031 in Figure !.5. in the case of lipids), will result in a repulsion of the other oxygen (021 in Figure 1.5.). In this way a conformational change in the phospholipid headgroup will be transferred in a re-orientation of the hydrophobic
region of tbe lipid molecule. As will be explained in Chapter 11, this re-orientation can induce cluster formation, which in turn may influence integral membrane proteins as ion channels, thus triggering them to open or to close. To get experimental support for this model, a number of vesicles bas been synthesized with different lipid composition. The
0 11 ..._". P· - N /
v,,,,,
o-/~o
'o
0ro
13
2 R 1 0)=o
R---Figure I. 5. The ext2•a negative aharge on the apieal paaition of the five eo-ordinated phospholipid intermediate results in repulsion of the oxygen bound via an P-D-G-C-0 sequenee.
the vesicle cell wall, using one and the same ionophore, is described in Chapter II to investigate several aspects of the theory.
1.3. Developments in organophosphorus chemistry. In the past few decades, research in organophosphorus chemistry bas developed enormously13. Study of the reactivity of model compounds has greatly enhanced the comprehension of the properties of five co-ordinated phosphorus compounds. So Westheimers studies14-17 on the hydrolysis of five-membered cyclic phosphonates have increased the understanding of the mechanistic aspects of phosphorylation reactions. It was found that the hydrolysis of five-membered ring phosphates as 1 in Figure 1.6.,
proceeds millions of times faster in comparison to acyclic phosphates as 4. This will apply for both ringopening (a) and exocyclic cleavage (b) of 1. In contrast, the cyclic
phosphonate 5 gave only very fast ring opening, no exocyclic hydrolysis. Westheiroer explained these observations on the
assumption, that the hydrolysis proceeds via a penta co-ordinated intermediate in a trigonal bipyramidal
configuration. Before these phenomena can be understood, some properties of five co-ordinated phosphorus compounds should be noted. As already described in chapter I.2. an important
o"o)
•H2o-(
110~
/OJ CH:lO - ; p ' o ~p HO 2 CH 0/ \0 3 0 0~/J
HO/ p\0 + CH30H EtO"-. /OEt 3 p ,~'"-. o o-4 0 00)
~<J
+ H20 11 CH30 - ; p CH3 0 5 HO 6Figure I.6. Cyclic and acyclic phosphates use« in the studies of West-heimer.
aspect of five co-ordination is, that the distribution of the ligands can not be spherically around the central atom, i.e. the ligands are not equivalent18. Two possible structures are favoured, as shown by X-ray analysis19-21: the trigonal
bipyramid (TBP) and the square pyramid (SP), shown in Figure I.7. In the TBP there are three equivalent equatorial and two axial honds, in the SP one axial and four basal honds.
Theoretical considerations based on MO and electrastatic calculations have predicted that the TBP is slightly more stable for acyclic penta co-ordinated phosphorus
a e ',
I
' p - ee#"
I
a TBP SP a: axial ligand b: basal ligand e: equatorial ligandFigure I.?. The trigonal bipyramidal (TBP) versus the square pyramidal(SP) configuration.
general an ideal TBP is seldom encountered, mostly a TBP is slightly distorted towards an SP geometry. In the TBP
configuration the axial bonds are longer and usually weaker than the equatorial bonds, a picture that can be ascribed by a pd-hybridization for the axial22-24 and a sp2-hybrid-ization for the equatorial bonds. However, the exact role of d-orbitals is still a subject of controversy25-27. Recent publications suggest a remarkable degree of s-character in the axial bonds of some radicals28-30. So the observed differences between ax,ial and equatorial sites in the TBP structure can, in a more differentiated picture, better be described by a substantially higher s-character for equator-ia! than for axial bonds. In addition, axial sites are preferred by electron withdrawing ligands, whereas electron donating ligands tend to occupy equatorial positions31. This polarity rule has been derived from many experimental
data32,33, and is supported by semi-empirica! calcula-tions34,35. Furthermore, it has been found that small rings usually span an axial and an equatorial position in the TBP configuration, due to the 90° angle between these two
bonds16. This is known as the strain rule. In fact, the presence of rings stahilizes this contiguration to such an extent, that most of the known stable phosphoranes contain one or more rings. One of the consequences of the differences in bond strength in a TBP is that leaving groups depart from the axial position16,34. Due to the microscopie reversibil-ity16, incoming nucleophiles also enter in the axis of the Tsp34. An aspect of five co-ordination which hampers
differentiation of axial and equatorial bonds in the TBP configuration, is the existence of pseudorotation.
Pseudorotatien for phosphorus compounds involves that the positions of the ligands are interconverted fast on NMR time-scale36-38. Several types of these 'permutational
isomerizations' are known, e.g. the Berry pseudorotation, in which two equatorial and both axial ligands change places39 via an intermediate SP contiguration (see Figure 1.8.).
/1 / I / / / :::::::t'P}--..::-. ~V /.,.l. ~· r<--- I( / / / V TBP SP TBP
ii'igure I.B. The Berry tien process.
The energy barrier for pseudorotation may be very low, especially if all ligands are identical18. However, if pseudorotations bring electron withdrawing ligands into equatorial positions34,40, or force small rings to span two sites of the same kind34, pseudorotatien will be severely hindered. Using the properties described above, the
experiments of Westheiroer (vide supra) are now readily explained (see Figure 1.9.). Initial attack of water on 1 yields intermediate 7. Subsequent proton transfer towards the endocyclic axial oxygen atom leads to formation of 8, resulting in 2 after ring opening (the axial P-0 bond is broken}. However, if the P(V) intermediate 1 undergoes ligand reorganization, 9 is formed. Upon leaving of the
axial protonated methoxy group, 3 is generated. The very fast rate of both processes is explained by the fact that cyclic, four co-ordinated phosphorus compounds are more strained than their acyclic analogues, whereas cyclic phosphorane
intermediates such as 7 or 9 are stabilized with respect to acyclic phophoranes. These factors lower the activation
7
Figure I.9. Pseudo-rotation in the experiments of Weetheimer.
enthalphy for hydralysis of cyclic compounds substantially41. Phosphonates as 5 can (see Figure 1.6.), after attack of water and prQton transfer to the axial oxygen, isomerize to either an intermediate with the ring carbon in an axial position (normally not occupied by electron donating groups), or to an intermediate with a di-equatorial five-membered ring, increasing the ring strain. These processes are energetically unfavourable and can not compete with ring opening, so no exocyclic hydralysis is found with the phosphonate .•
More recently, many other stereochemical and kinetic data in phosphorylation reactions and in group transfer reactions have been rationalized by invoking phosphorane intermediates, including the group transfer reactions in tricyclic 'caged' phosphatranes by Van Aken et al~2,43.
1.4. Transfer of conformational changes in a phosphate group to the hydrophobic part of organic molecules. In the phosphorylation reactions of Westheimer, substitution was achieved at phosphorus, whereby both incoming and leaving groups entered and departed via the axis of an P(V) TBP. In biochemistry a lot of phosphate groups are temporarily activated without breaking the honds with the rest of the biomolecule. In this case incoming and leaving group are the same molecule, e.g. water. Dependent on the life time of the P(V) TBP intermediate the activated system will be able to
relax to a lower energy state. Another configuration will he occupied, that is better able to accommodate the new charge distribution over the ligands around phosphorus. Usually this will happen by turning away one electronegative part of the molecule from the other. Theoretical verification of charge
0 11 . P, +LH Ho~;!
'o-
....
Hs';.;.<'Os·,....
Hs"~jo
H~,·net atomie P(IV) charge ( e. u.) 0 ( 1 I) -0.267 0(5') -0.289 p +0.370 L ... P(V)H P(V) r.=HNMe L=OH L=HNMe L=OH
-0.274 -0.274 -0.291 -0.296 -0.316 -0.318 -0.348 -0.355 +0.340 +0.348 +0.409 +0. 451 -0.031 -0.133 -0.320 -0.436
1.10. Charge distribution on the ligands of a tetrahydrofurfuryl model system ~hen going from a four to a five co-ordinated intermediate;
enhancement on apical ligands in a P(V) TBP model compound for DNA was recently published by van Lier et alJ1. The net atomie charge on various atoms in the tetrahydrofurfuryl model system and its P(V) TBP counterpart are given in Figure
!.10. Experimental evidence for the rotation, resulting from this charge enhancement, was very recently given by Koole et alJ2. They synthesized a number of four and five co-ordinated mutually resembling phosphorus model compounds and found a significantly greater population of the gauche(-) conforma-tion for axially situated tetrahydrofurfuryls around the C4'-Cs' bond in the 5' P(V) TBP tetrahydrofurfuryls with respecttotheir related P(IV) compounds. In Figure !.11., some of the model compounds used are given12.
L:: Ph,OEt Y:O,CH2
L:Ph,OEt
Y = O,CH2
Figure I.ll. Four and five ao-ordinated model compounds with different Zi-gands substituted on the phosphate group and in the tetrahydPofUrfuryZ ring.
The Newman projectionsof the rotamers around the C4'-Cs' bond are given in Figure I.12. The rotamer populations x(g+), x(gt) and x(g-) could be determined from the time-averaged coupling-constants Ja4'as' and Ja4'as"12. In the
P(IV) compounds Os' and Y are oriented cis to each other (the gauche(+) or the gauche(t) rotamer) in the case Y
=
o,
due to the gauche effect44. This effect is defined45 as theOs· H s" Hs·
Y*C,
Y*C,•
Y*C,·
Hs. Hs" Os• H5• H5 05 •
H •. H •• H ••
g• gt
g-I.l2. Newman around the P-0-C-C-0 sequence.
number of gauche interactions between the adjacent electron pairs and/or polar bands" and originates from bond-antibond interactions45. The only compound that differs substantially possesses a CH2 group on the Y position (see Figure !.11.) and the C4'-Cs' rotaroer distribution is consequently not dominated by the gauche effect. In the P(V) compounds Os' and 01' are orientated more transtoeach other, i.e. the g
population is enhanced, due to the repulsion of two more negatively charged oxygens. Only the compounds with Y = CH2 show no difference in population with the four co-ordinated intermediate, which is clearly the consequence of the
P-0-C-C-C sequence present in the molecule, instead of the P-0-C-C-O sequence, that is responsible for repulsion.
References and notes.
1. R. Harrison and G.G. Lunt, 'Biological Membranes, Their Structure and Function', Blackie, Glasgow, 1980; p 62. 2. S.J. Singer and G.L. Nicolson, Science 1972, 175,
720-731.
3. A. Tardieu, V. Luzzati and F.C. Reman, J. Mol. Biol. 1973, 75, 711-733.
4. F.T. Presti, R.J. Pace and S.I. Chan, Biochemietry 1982, 21, 3831-3835.
5. A.G. Lee, Biochim. Biophys. Acta 1977, 472, 237-281. 6. A. Blume, R.J. Wittebort, S.K. Das Gupta and R.G.
Griffin, Biochemietry 1982, 21, 6243-6253. 7. In reference 1, p 119.
8. In ref~ence 1, p 12. 9. In refere~e l, p 87. 10. In reference 1, p 86.
11. J.J.C. van Lier, L.B. Koole and H.M. Buck, Reel. Trav. Chim. Pays-Bas 1983, 102, 148-154.
12. L.B. Koole, E.J. Lanters and H.M. Buck, J. Am. Chem. Soc. 1984, 106, 5451-5457.
13. For up-to-date reviews on the subject, see the series 'Organophosphorus Chemistry' (Specialist Periodical Reports), S. Trippett, ed., The Chemical Society, London.
14. F. Covitz and F.H. Westheimer, J. Am. Chem. Soc. 1963, 85, 1773-1777.
15. E.A. Dennis and F.H. Westheimer, J. Am. Chem. Soc. 1966, 88, 3431-3433.
16. F.H. Westheimer, Acc. Chem. Res. 1968, 1, 70-78.
17. R. Kluger, F. Covitz, E. Dennis, L.D. Williams and F.H. Westheimer, J. Am. Chem. Soc. 1969, 91, 6066-6072. 18. R. Luckenbach, 'Dynamic Stereochemietry of
Pentaco-ordinated Phosphorus and Related Elements', G. Thieme, Stuttgart, 1973.
19. B.L. Muetterties and R.A. Schunn, Quart. Rev. Chem. Soc. 1966, 20, 245-299.
20. T.E. Clark, R.O. Day and R.R. Holmes, Inorg. Chem. 1979, 18, 1653-1659.
21. T.E. Clark, R.O. Day and R.R. Holmes, Inorg. Chem. 1979, 18, 1668-1674.
22. R.F. Hudson and M. Green, Angew. Chem. 1963, 75, 47-56. 23. A.J. Kirby and S.G. Warren, 'The Organic Chemistry of
Phosphorus', Elsevier Publ. Co., Amsterdam, 1967. 24. F. Ramirez, S. Pfohl, E.A. Tsolis, J.F. Pilot, C.P.S.
Smith, I. Ogi, D. Marquarding, P. Gillespie and P. Hoffman, Phosphorus, 1971, 1, 1-16.
25. O.A. Bochvar, N.P. Gambaryan and L.M. Epshtein, Russ. Chem. Rev. 1976, 45, 660-670.
26. T.A. Halgren, L.O. Brown, D.A. Kleier and W.N. Lipscomb, J. Am. Chem. Soc. 1977, 99, 6793-6806.
27. M.A. Ratner and J.R. Sabin, J. Am. Chem. Soc. 1977, 99, 3954-3960.
28. J.H.H. Hamerlinck, Ph. D. Thesis, Eindhoven Oniversity of Technology, 1982.
29. J.H.H. Hamerlinck, P. Schipper and H.M. Buck, J. Org. Chem. 1983, 48, 306-308.
30. J.H.H. Hamerlinck, P. Schipper and H.M. Buck, J. Am.
Chem. Soc. 1983, 105, 385-395.
31. P. Gillespie, P. Hoffmann, H. Klusacek, D. Marquarding,
s.
Pfohl, F. Ramirez, E.A. Tsolis and I. Ugi, Angew. Chem. 1971, 83, 691-721.32. E.L. Muetterties, W. Mahler and R. Schmutzler, Inorg. Chem., 1963, 2, 613-618.
33. E.L. Muetterties, K.J. Packer and R. Schmutzler, Inorg. Chem. 1964, 3,1298-1303.
34. D. Marquarding, F. Ramirez, I. Ogi and P. Gillespie, Angew. Chem. 1964, 85, 99-127.
35. F. Keiland
w.
Kutzelnigg, J. Am. Chem. Soc. 1975, 97, 3623-3632.36. E.L. Muetterties, J. Am. Chem. Soc. 1969, 91, 1636-1643. 37. E.L. Muetterties, J. Am. Chem. Soc. 1969, 91 , 4115-4122. 38. J. I. Musher, J. Chem. Educ. 1974, 51 , 94-97.
40. I. Ugi and F. Ramirez, Chem. Br. 1972, 8, 198-210. 41. J.A. Gerlt, F.H. Westheiroer and J.M. Sturtevant, J.
Biol. Chem. 1975, 250, 5059-5067.
42. D. van Aken, l.I. Merkelbach, A.S. Koster and H.M. Buck, J. Chem. Soc., Chem. Comm., 1980, 1045-1046.
43. D. van Aken, l.I. Merkelbach, J.H.H. Hamerlinck, P. Schipper and H.M. Buck, A.C.S. Symp. Ser., 1981, 171, 439-442.
44.
s.
Wolfe, Acc. Chem. Res. 1972, 5, 102-111.45. T.K. Brunck and F. Weinhold, J. Am. Chem. Soc. 1979, 101, 1700-1709.
II. Sodium ion transport across biologica! membranes.
I I . 1. Theory.
II.l.l. Cluster formation in biologica! membranes induced via a phospholipid P{V) TBP intermediate.
In Chapter I the principle of induction of an electron flux into the axis of a five co-ordinated phosphorus (P(V)) trigonal bipyramidal (TBP) intermediate was discussed. In those model compounds, the extra negative charge on the axial oxygen (Os') in the P(V) TBP intermediate resulted in repulsion of another oxygen (01 '), bound via an
o-e-c-o
sequence to the phosphate group, and located in a tetrahydro-furfuryl ring (see Figure II.l(a)). -(a) I. .1' I'
I bl .1•'
'
Figure II.l. Repulsion between the two oxygens in a
P-0-c-c-o
seque~e as a aonsequenoe of thè transition from a four ao-ordinated to a five ao-ordinated intermediate in (a) a tetrahydrofurfurylphosphate and (b) a phospholipid.In this Chapter, an attempt will be made to show that the same process can occur in phospholipids, and that this
process could be the 'trigger' to activate proteins, such as ion channels, emeedded in a lipid bilayer matrix via uptakè in clusters 1. In Figure II •. l (b) one can distinguish the same
o-e-c-o
sequence, bound at the axial position of a phosphate group, as discussed for the model compounds in Chapter I. Repulsion between the two oxygens 021 and 0312 of the P-0-C-C-0 sequence will cause a shift of the sn-2 chain in the direction perpendicular to the bilayer surface (see Figure II.1.(b)). However, the model compound& as (a) in Figure II.1. are monomers, dissolved in organic solvents, and thus able to re-orientate freely in solution. The phospho-lipids, on the contrary, are built in in the lipid bilayer, with their long hydracarbon chains interacting via 'van der Waals' interactions with the neighbouring chains. So the shift of the hydracarbon chains along each other willnormally take a high energy barrier to overcome. A plausible adaptation of the bilayer by which this process can be aided, is accompanied by a change in the angle of tilt of the hydra-carbon chains to the bilayer normal. Phase diagrams of
phospholipids show, dependent on temperature and percentage water or different lipid, several one and two phase regions3, in which among others the angle of tilt to the bilayer normal varies. In a special temperature interval, the phase transi-tion region, ranging from the main phase transitransi-tion tempera-ture down to a temperatempera-ture around the pretransition4,5, smal! domains of different density (and thus different angle of tilt) occur next to each other. Such clusters are reported for mixtures of phosphatidylcholines (PC) with phosphatidyl-ethanolamines (PE)3, cholesterol6 or proteins7. The co-oper-ative change in the angle of tilt of all the lipid molecules in the same cluster, can minimize the energy barrier that has to be overcome. Although there is controversy about the exact nature of the pretransition, a continuous change in the angle of tilt is always included in the description. Some authors conclude that the angle of tilt will change from tilted at
the pretranaition temperature to parallel to the bilayer nor-mal at the main phase transition8. Others believe the angle of t i l t reaches a local minimum at the pretransition, accom-panied by a transition from a tilted conformation, via a tilted and rippled two dimensional structure9 to a one dimen-sional structure with the chains perpendicular to the bilayer surface. Experimental evidence for a variation in angle of t i l t of the hydracarbon chains accompanying hydracarbon chain shift was given by Blume3 and Chen10. Comparison of 13c and 2H NMR spectra of phospholipids, labelled respectively with 13c at the sn-2 carbonyl group3 and with 2H at the 4-position of the same sn-2 chain, suggests that a conformational change of the carbonylgroup precedes chain melting on increasing temperature. This could be an indication of constantly
developing P(V) TBP intermediates, meeting below the
pretran-lipid MMPC MPPc(a) MSPc(b) PMPC PPPC PSPC SMPC SPPC SSPC ma in transition temperature 23.6 35.1 38.6 27.3 41.1 49.0 29.4 43.9 54.2 pretransition temper at ure 14.4 22.8 10.8 34.8 39.9 20.0 30.8 50.4
(a) MPPC
=
a myristoyl chain (M) bound at the sn-1 position and a palmitoyl chain (P) bound at the sn-2 position of the phosphatidylcholine (PC) glycerol backbone.(b) S
=
a stearoyl chain.Table II.l. Main transition and pretransition temperature as a function of the chain Zength of the chain at the sn-1 and the sn-2 position of phosphatidyZchoZine.
sition temperaturel an energy barrier too high to transmit the repulsion between the two oxygens of the
o-e-c-o
sequence in a shift of the sn-2 chain. The activation around the pre-transition temperature is high enough, howe.ver, to induce a conformational change at the carbonyl-group next to 021• At higher temperatures an ever greater part of one and the same sn-2 chain and/or an ever greater part of the total number of sn-2 ebains will be aetivated, untill at the main phase transition all the ebains are oriented perpendieular to the membrane surfaee. Moreover, the pretransition behaviour shows strong dependenee on eomposition10. PCs with myristoyl(C14), palmitoyl (C16) and stearoyl ebains (C1s> at the sn-1 and/or sn-2 position meet a higher energy barrier to melt if the sn-2 chain is longer (see Table 11.1). Another environ-mental constraint is met in the headgroup region (see Figure
II.2.).
---H....,_./
H 0-o
1/,,,, •..~
- o
-o
""=-k-J-, . 06 -;:N / 3
~
=
~
l L1' I'
Figure II.2. Ringformation in the phosphatiQY~eho~ine headgroup upon formation of a five ao-oPdinated intermediate.
In the headgroup, accommodation of a fifth ligand to form a five eo-ordinated intermediate will cause re-orientation of
the ligands around phosphorus. A lot of phospholipid
molecules contain zwitterionic headgroups (see Figure II.3.), that are arranged with alternating charge in the bilayer11. This model of intermolecular interaction between e.g. the N-methyl protons of one PC molecule and the phosphate of a
neighbouring PC molecule, however, still allows for
considerable freedom of movement about the various honds in the headgroup11.
phosphatidyl-choline
II.3.
sphingomyelin phosphatidyl- phosphatidyl-ethanolamine serine
w·ith a zwittericm:c
Thus formation of a five co-ordinated phosphorus intermediate must be accompanied by re-organization of the charge in the total bilayer. This can occur, for example, by intrarnolecular compensation of the charge12, thus creating a more or less neutral molecule, or, at a physiological level, by de- and adsorbtion of mono- and di-valent cations13,14.
Intramolecular compensation of charge can be established by pseudo-ringformation, in which positively and negatively charged groups are brought close to each other12 (see Figure II.4.). Here, another aspect of the pretransition behaviour is met, the relative cross-sectional areas of headgroup and chain region. In molecules as PC, the headgroup in
excluded cross-sectional area tban the lipid bydrocarbon cbains. Tbe ebains adopt a tilted conformation to fill .in a potential void in tbe bydrocarbon cbain region6. Above tbe pretransition temperature, an increasing number of ebains orient more perpendicular to the membrane surface8,9, so that the excluded cross-sectional area of tbe .headgroup must have been diminished. Tbis is confirmed by tbe observationlS tbat PE, N-methyl and N,N-dimethyl PE do not exhibit such pretransition behaviour, since the cross-sectional areas of their headgroups are smaller. The effective cross-sectional area of the PC headgroup can be diminished further, after di-equatorial16 pseudo-ringformation and pass down of the charge, by pseudo-rotation, through which the pseudo six-membered ring will be orientated temporarily
axial-equator-ial. This pseudorotated structure will, at the same time, prevent electron back donation to the fifth ligand, e.g. water, and return of the intermediate to the four co-ordi-nated state. De- and adsorption of mono- and divalent cations can complete the picture sketched above. During the physio-logical process of the excitation of an axon, for example, momentary desorption of ca2+ ions from the outer monolayer of the membrane is reported13,14 upon activation of the axon, followed by adsorption of monovalent ions as Na+. This decrease in positive surface-bound charge of cations can dfminish repulsive forces, intended to keep the headgroup
'stretched' in the unactivated axon17.
Finally, again in the example of the excitation of the axon, the potential at rest is negative inside the axon, pulling the positively charged end of the stretched headgroup -N(CHJ)3+ of the outer monolayer phospholipids into the membranes. During activation of the axon, a positive poten-tial inside17 will push the positive charged end of the head-group outwards, enabling the headhead-group to re-orientate. Thus, a set of environmental physiological conditions is realized, enabling a P(V) intermediate to develop and to pass the information, stored in its renewed charge-distribution, down to the hydracarbon region, for the case of the motionally
restricted phospholipid molecules. On a molecular level this processcan be summarized as follows (see Figure II.4.). ca2+ ions desorb, and are replaced by Na+ ions. The internal potential is changing from negative to positive. As a conse-quence the headgroup is no langer forced in an extended and inward pulled conformation and gets the opportunity to
re-orientate. The always existing P(IV) ~ P(V) equilibrium, under 'resting' conditions laying at the side of the P(IV)
Figure II.4. The in the physioîogicaî conditions accompanying
the transition of a four to a f"ÎVe co-oràinated intermediate.
compounds, will be shifted in the direction of the P(V) intermediate, since this conformation now is stabilized by the formation of a di-equatorial16 pseudo-six roerobered ring. The positively charged nitrogen of the choline headgroup shields the negative charge of the formerly double bonded oxygen, thus polarizing the P=O bond, by which the electro-philicity of the phosphorus atom will be increased18. This process will be promoted by nucleophilic attack of e.g. a water molecule19, normally present in excess in the headgroup
layer of the membrane, thus generating the P(V) TBP inter-mediate. The decrease in cross-sectional area of the head-group due to ring formation, will cause decrease in the angle of tilt of the hydracarbon chains. The increased electron density on the axial oxygen of the phosphate group will
induce repulsion of 021• bound via an o-e-c-o sequence to the phosphate group. This process will aid, or maybe it is the main cause, of a further decrease in the angle of tilt of the hydracarbon chains, through a 'shift' of the hydracarbon chains along each other. The difference in effective chain length10, 20-22 is diminished. Co-operative change in the angle of tilt of a number of phospholipid molecules is needed to maximize the 'van der Waals' interactions between neigh-bouring acyl chains. This will lead at a macromolecular level to formation of a cluster with an average angle of tilt differing from the surrounding matrix. The relaxation time of such a cluster is appreciably greater than for other charac-teristic movements of the molecule23. So the short-living P(V) TBP intermediate initiatea the formation of a cluster with a much longer life-time, through which a time scale can be reached at which physiological processes can take place23. Although the physiological conditions of the above process are borrowed from the excitation of an axon, one can imagine the same conditions for other membranes. Over most membranes an ion-gradient is maintained by ion pumps, so a potentlal exists over most membranes. Divalent ions as ca2+ and Mg2+ are bound to most membrane surfaces to an extent dependent on the physical state of the lipids23. Mostly they are bound more strongly than monovalent ions such as Na+ and K+, that are present in all intra and extra-cellular spaces. The local oircumstances may change, a P(V) TBP intermediate can be built up under several sets of conditions.
11.1.2. Activation of membrane proteins by uptake in phospholipid clusters
A break in the Arrhenius plot of ATP-ase activity versus the reciprocal temperature has been reported in dioleoyllecithin substituted ATP-ase, when reaching the temperature of cluster formation24. A transition in the temperature dependenee çf ca2+ accumulation25 in Sarcoplasmic reticulum membranes is attributed to a change in entropy of activatien rather than to the free energy of activation. Results that are consistent with an order-disorder transition invalving the lipid alkyl chains25. Computer simulation studies concerning phase separation in lipid bilayers containing integral proteins7, show a system that separates into an essentially pure lipid phase and a protein-rich phase containing melted lipids between Tk and Tc· Here Tk is the melting point of clusters, and Tc is the {main) melting point of lipids. Addition of oleic acid to a lipid deficient membrane26 produces a fluid membrane structure, which is most likely an essential
requirement for the reconstitution of the calcium dependent ATP-ase activity. Addition of stearic acid, on the contrary, has no activating effect on the calcium dependent ATP-ase26 and creates a gel-like lipid structure. From the above-mentioned and other27-29 articles, it becomes clear that a certain fluidity is essential for the activatien of membrane proteins, and that this fluidity is reached in a temperature
range in which cluster formation appears.
An often encountered objection against the relation between cluster-formation and protein activation is, that gel state lipids do not appear to be present in most biologica!
membranes30. However, this is only partly true. Harrison and Lunt conclude31 that, although hydracarbon ebains in natural membranes are believed to be generally in a fluid state at physiological temperatures, the presence of sterals and proteins may lead to local variation of the mobility in the membrane. Moreover, the degree of lateral phase separation is believed to be influenced by a number of external factors,
e.g. water content31, proton and cation concentration32, ionic strength32 and potential fiela19. Under influence o.f the above-mentioned factors, cluster-formation is believed to persist far above the main phase transition temperature of a pure lipid mixture.
Also pore-mediated ion transport shows some peculiar
characteristics while planar bilayer membranes pass the phase transition region o.n heating33. Planar bilayers consisting of mixed-chain lipids and modified by pore-forming antibiotica as Gramicidin A, do not show any peculiar effect on Tc, the main phase transition temperature (29°C). Bowever, at 22-23°C a pronounced maximum in pore-induced conductance is seen. The effects observed are interpreted in terros of lateral -phase separation into pure lipid and lipid-antibiotic domains33. Consequently, the polypeptide Gramicidin A is an ideal model for the ion-channel forming proteins, obeying the same
temperature dependenee of protein activation upon cluster formation. A schematic representation of Gramicidin A34 is given in Figure II.5.
0 N
0 0
• H ...,.. H- bond
Figure II.5. The Gramicidin channel oomprises ~o polypeptide ahains in
On a molecular level, protein activation upon uptake of the protein in a cluster, can be described as follows. An
integral protein in a fluid environment (the cluster} could be free to adopt the tertiary structure necessary to function as an ion-channel. A gel-like matrix can displace some
special group of the channel-forming protein out of its critical position35 and/or disturb a protein in helix form36. For the sodium ion-channels of myelinated axons a model is given in which four energy harriers in the pore cernprise the selectivity filter in the ion-channet35. These four harriers consist of dehydration and hydration steps, enabling a
partially dehydrated sodium ion37 to pass a narrow gap besides a strongly co-ordinating carboxylic acid group38. Completely hydrated, the sodium ion will not be able to pass the narrow selectivity filter of 3 x 5 Ä. A very small
displacement of the carboxylic acid group can make the ion-channels impermeable.
2
Figure II.6, The of the ion channeL.
In
energy harriers comprising the selectivity filter
Although the correlation between a fluid lipid environment and activation of proteins is suggested by quite a number of authors (vide supra), this is not necessarily a general
principle. Also the reverse process, i.e. protein activation upon uptake in a gel-like cluster, is a process that should not be neglected. In this way a number of different proteins could be activated in succession, if e.g. the potentlal is constantly changing from negative to positive and a whole scala of states of different rigidity is passed through. Finally, support for the above-mentioned theory is given by the fact, that 2-amido PC, contrary to PC, is found to be inhibitory for integral proteins39,40 (see Figure II.7.). The oxygen esterified to c2 of the glycerol backbone of PC is essential for the transfer of conformational change in the headgroup towards the change of tilt of the hydracarbon chains. If this oxygen is replaced by the less
electro-negative nitrogen of 2-amido PC, less repulsion and resulting acyl chain shift will be expected. Moreover, the hydrogen bridge found in X-ray analyses of comparable lipids41, will hinder acyl chain shift and headgroup re-orientation (Figure
11.7.).
)(
Figure II.7. Hindered repulsion and aeyZ ahain shift in 2-amido phospha-tidyZehoZine.
II.2. Experiments.
11.2.1. Introduction.
The characteristic feature of the model, described in this Chapter, is that changes in the lipid environment of a
protein are the 'trigger' for the protein to be activated (to place some particular functional group just in or just out of the right position). Other authors propose a mechanism in which changes in membrane potential, pH, ionic strength etc., directly influence the channel-forming protein (in the case of ion-transport) to open, a mechanism that developed under the influence of experiments with the ion channel blockers tetrodotoxin42 and saxitoxin43.
To get a more decisive answer about this question, the
experimental conditions of the investigations described below are chosen so, that only the lipid composition of the mem-branes varies, leaving the concentratien of ion channels, ions, probes and buffers, as well as temperature, as constant as possible. Vesicles are formed with a diameter of approxi-mately 1000 Á, their wall existing of one double layer of lipids. As a reference, vesicles of egg yolk lecithin are chosen, to which 10 to 50% of synthetic or natural, specific lipid is added, to vary the total lipid composition. Attempts to make vesicles of one well-defined synthetic lipid as
reference, failed, since the temperature of formation of the vesicles had to be above the main phase transition tempera-ture, Tc. Tc will vary from 24•c for dimyristoyl (C14l, via 41•c for dipalmitoyl (C16l to ss•c for distearoyl (C1al phos-phatidylcholine44. The last two temperatures where too high to be constantly maintained throughout the whole procedure of synthesis. Since Tc of egg yolk lecithin is around o•c, the choice of this lipid experimentally gave no problems.
As model for the ion-channel protein, the ionophore
Gramicidin A was chosen for a number of reasons, in addition tothese mentioned in Chapter 11.1.2. Gramicidin A is a pore-former, specific for sodium ions. 1t is a pore-former and not
a carrier, so it builds up a permanent channel comparable to natura! ion-channels, and does not diffuse through the
membrane as carriers do. A pore-former also functions below the main phase transition temperature so that the temperature range in which it is active is greater, while the conductance of carriers falls below Tc to the state of bare membrane conductance (not pore mediated)33,45. The spontaneous
current fluctuations observed with unmodified planar bilayers near the lipid phase transition temperature, containing a few molecules of Gramicidin A, reminds of the idea of 'cluster-activation' of channela46, i.e. Gramicidin A is activated if it is taken up in such a cluster. Formation of a cluster around a Gramicidin A molecule activatea the channel to open. The channel stays open during the life-time of a cluster, that can change randomly, but the conductance reached is always the same, unless the cluster will decay before the maximum conductance is reached.
A Gramicidin A channel is formed by association of two poly-petides at their N-formyl ends. Each chain is folded into a B helix, which resembles a rolled-up B pleated sheet (see (a)
in Figure 11.8.).
Figure II. 8. Four possib Ze confoi'mations of t;he Gramicidin channe L
,--~
Finally, Gramicidin A incorporates spontaneously in the
veeiele wall after addition, since its amino acid sequence is one of the most hydrophobic ones known47.
' " (el 1=2410 (I) I 11 1=11.0 00 '
"
[d) t=13 30 (k) I 11 (c) 1=458 ( j) (i) ( b) t = 0 I 00 11 ( h) (g) (a) I 11 t = 0 00 ( f )Figure II.9. 23Na Nii!R spectra (79.4 MHz) of a dispersion of vesicles of
egg yolk phosphatidylcholine plus 10% sphingomyelin (a) at t=O before addition of the probe, (b) at t=O after addition of the probe, (c)-(l) after addition of Gramicidin; T = 25°C.
Moreover the exterior of the channel consists again of the most hydrophobic parts of the peptide48, thus creating an oxygen lined, probably water-filled, central channel.
Gramicidin A is added in last resort to a buffered solution with approximately 1000
A
diameter vesicles, around which the NaCl solution is replaced by LiCl by means of ultrafiltra-tion. Thus a sodium ion gradient is formed of about 102, the equilibration of which is started at the moment Gramicidin A is added. A shift reagent49, Dy I N(CH2C02)3J23- has been used to distinguish between 23Na+ inside and outside the vesicles.II.2.2. Results.
A homogeneous suspension of 1000 Ä diameter, single bilayer phospholipid vesicles is prepared by the procedure of Enoch and Strittmatter50. The solubility and nature of the surfac-tant51 deoxycholate, together with its concentration50 (a phospholipid to deoxycholate molar ratio of 2:1 is used), determine the diameter of the resulting vesicles. After formation of the vesicles, the detergent is removed to make the vesicles impermeable to sodium ions, next external NaCl is replaced by LiCl by means of succeeding ultrafiltration steps. After this stage there is 100 mM NaCl inside the vesicles and the aqueous space outside the vesicles is 50 mM in LiCl and < 1 mM in NaCl. The 23Na NMR spectrum (79,4 MHz, Bruker CXP-300) of a dispersion of vesicles consisting of egg yolk lecithin +10% sphingomyelin is given in Figure II.9.a, the single sharp resonance at 1802 Hz ± 2 Bz repreaenting both the Na+ inside and outside the vesicles. Fig. II.9.b shows the spectrum after the outside aqueous space is made 3 mM in triethanolamine dysprosium nitrilotriacetate
[HN(CH2CH20H)3)3Dy[N(CH2C02)3]2, according to the metbod of Pike et al49. The single resonance of Figure II.9.a is split into two peaks, the smaller one49 65 ± 10 Hz upfield from the peak position of Figure II.9.a repreaenting Na+ outside the vesicle and the large one 4 ± 1 Hz downfield repreaenting Na+
1.0 PC trom 0.9 egg yol k 0.8 6 + 50 •;. OMPC 7 • so·~ OPPC 0.7 R + , oo;. OPPC
1
5.
10% OSPC 0.6 3 • 10% OMPC 0.5 0.4 2 + 10% sphingo-mye I in 0.3 9 •10% P inOSitOI 10 + 1 0°/o OPPE PC purefro m egg yol k
0.2 0.1 8 + 10o/o P serine 6 3 5 - t f h r l
Figure II.10. The time of the re~ative integra~s of the two
peaks of Figure II.9. ten different differing in ~ipid
inside, the absolute magnitude of the shift being dependent on the exact concentratien of the probe49. The fraction of the total integral, due to the inside resonance varied from 0.77 to 0.92 for the various samples. The value for the fraction of the total aqueous volume inside the vesicles is calculated, assuming an internal volume of 2960 ml per mole of lipid52 and a final lipid concentratien of 10 mM, to be 0.05. There could be some error in this ratio because of inaccuracy in the knowledge of the final lipid concentration. Bowever, other studies using the method of Enoch and Stritt-matterSO, where phosphate analyses was conducted on the final solutions, were in good agreement with the above-mentioned fraction of lipid left after synthesis of the vesicles. Combination of these numbers with 100 mM Na+in• yields a Na+out concentratien of 0.27 mM in the case the fraction of the total integral due to inside resonance at t = 0 is 0.92 or 0.92 mM in the case this fraction is 0.77. Since any 1eakiness of the vesicles would affect the observed ratio, all starting spectra were obtained between 0.5 and 3 hrs after the last ultrafiltratien step, and samples with a fraction due to inside resonance smaller than 0.77 were not used further. E.g. for the sample of egg yolk lecithin with 50% DSPC it was not possible, even after several attempts, to obtain a sample with a fraction due to internal resonance greater than 0.30. Immediately after the spectrum of sample + probe was obtained, the salution was made 0.04 pM in the ionophore Gramicidin53-55. This amounts to ca. 3 Gramicidin channels per vesicle49 and induces a rapid efflux of Na+ down its ccncentration gradient, as can be seen in the Figures II.9.c-l. They depiet some of the spectra obtained and show the time evolution of the spectrum measured in minutes from the time of addition of Gramicidin, for a sample displaying an intermediate time course in the total series measured (egg yolk + 10% spingomyelin). The spectra given are power spectra in which the integral is proportional to the square of the number of the sodium nuclei56. The power spectra are taken to cancel the influence of the phase-correction, that will vary
with time during the period the automated measurements are recorded. A plot of the logarithm of ratio R, of the frac-tional integral (inner/total) at t = t to the fractional integral at t
=
0, against time is shown in Figure II.10. The time during which the spectra were recorded after addition of Gramicidin varied from 2 to 13 hrs, dependent on how fast theion transport took place. For 50% DPPC not the total time-course is given, since between 5 and 13 hrs after addition of Gramicidin the slope of the plot was identical to that of the part shown between 0 and 5 hrs.
II.2.3. Discussion.
The efflux of Na+ ions shows at least two stages49. Directly after addition of Gramicidin a passive one-for-one Na+ for Li+ exchange out of and into the vesicles takes place, both
ions moving down their concentratien gradients, although the cl- transport may play a role at this stage (vide infra}. This stage will end when the Li+ gradient is dissipated
(47.5 mM both inside and outside). Since the Na+ gradient still exists at this point (52.5 mM inside, : 3 mM outside,
R = 0.52), during the second stage the Na+ gradient will be further dissipated at the expense of creating a new Li+ gradient, still through a one-for-one exchange. This stage will end when the ratio of inside concentratien to outside concentratien has the same value for both Na+ and Li+, so that the same diffusion potential for both ions is reached. Here Na+ in is 10 mM, Na+ out is 5.3 mM, Li+ in is 90 mM
and Li+ out is 45.3 mM, the fraction of total Na+ inside is 0.09. True equilibrium will only be obtained after a third stage, namely passive nonfacilitated cl- transport out of the vesicles. This stage will end when the fraction of total Na+ inside is equal to the analogous volume fraction, 0.05. In these rough calculations osmotic swelling, a possible pH gradient due to permeation of the counter ion of the probe HN(CH2CH20H)3+ and the Donnan effect caused by the impermeant
Dy[N(CH2C02)3l23- ar~ ignorea50. The transitions of the curves, shown in Figure II.10, are located between R = 0.5 and R = 0.3, the transitions being at lower R if ion trans-port is faster. This could be an indication of leakage before Gramicidin is added, although measurements of ten different blancos (after addition of the probe) of the same sample in a time period of half an hour showed no significant decrease of the ratio of Na+in to Na+out•
The fast process between R = 1 and R
=
0.5, corresponding to the one-for-one exchange of Na+ and Li+ down to theirgradient, is believed to be limited by the Gramicidin induced Li+ transport, which is ca. 1/6th as fast as that of Na+ 57. Thus the slow process, below ca. R
=
0.5, would correspond to the essentially simultaneous occurrence of the second and third stages (vide supra) implying that they have very simi-lar permeability constants. This is supported by measurements of permeability coefficients of pass!ve nonfacilitated trans~port of cl- 52. So both stages befare and after R
=
0.5, are involved in sodium ion transport.As can beseen in Figure II.10, the relative sequence in velocity of ion transport for the various samples is not interchanged when passing R
=
0.5.The reference sample, phosphatidylcholine from egg yolk exists of predominantly C16 (34.3%) and C1a chains (59.8% C18:0• C18:1 and c18:2)58, as can beseen in Table
II.2. Ion transport over a vesicle cell wall consisting of egg yolk phosphatidylcholine is relatively fast, compared to most of the other samples (2-10 in Figure II.10.). Clearly, the relative quantities of different chain lengtbs and
saturation is such, that the rigidity of the matrix is ideal to accommodate for the chain shift that results from the
formation of a five co-ordinated phosphorus intermediate. Increasing the percentage of synthetic saturated ebains
(samples 3-6), the matrix will adopt a more gel-like charac-ter, in which the packing of the optimally ordered ebains is tighter, so the chain shift is more difficult. When
becomes slower, although the difference between ~he sample with 10% DSPC and with 10% DPPC is too smal!, compared to the deviation in slope due to measuring faults, to be called significant. Addition of more (50%)'of the same saturated chains again gives slower ion transport, in which the slopes of the plots of 50% DMPC and 50% DPPC are indistinguisable. Addition of 10% of sphingomyelin from egg yolk, containing primarily saturated palmitic acid chains at the sn-2 position results in ion transport that is slower than that of the reference sample of phosphatidylcholine from egg yolk (1), but faster than that of the samples 3-6. The slower ion transport of sample 2 compared to sample 1 could stem from the restricted repulsion of the sn-2 nitrogen (see Figure II.11.) through which the sn-1 chain is bound to the glycerol backbone. The significantly faster ion transport compared to the samples 3-6 can be explained by the fact that the sn-1 chain is significantly shorter (two atoms less than in DPPC, viz. -CH=CH-(CH2l12-CH3 directly bound to the glycerol back-bene, and the appearance of a double bond), thus creating a molecule comparable to MPPC (see Table II.1.).
Moreover, addition of a clearly different lipid will enhance the heterogenity of the matrix, making the matrix more fluid, and enabling lipids other than sphingomyelin to translate a five co-ordinated phosphorus intermediate more easily in a hydrocarbon chain shift.
Samples 8, 9 and 10 contain additions with primarily C16 or C18:0 to C18:2 (see Table II.2.) as lipid alkyl chains, thus changing the overall fluidity of the matrix not too much, compared to 1, on additions of 10%. Egg yolk phosphati-dylcholine with 10% phosphatidylserine shows considerably faster ion transport compared to the reference sample. The availibility of two ligands within the serine, one as fifth ligand to build up a five co-ordinated intermediate and one to polarize the original P = 0 bond, could cause an appreci-ably increased life-time of the five co-ordinated inter-mediate, which explains the fast ion transport observed (see Figure I I. 12.).
chain length sphingomyelin phosphatidyl-and choline S-0756 ethanolamine inositol serine P-8518 saturation P-6013 P-4513 P-5766 C14:0 0.1% 0.6%
-
-
-c16:0 34.3% 86.2% 23.6% 32.8% -c1s:o 12.0% 6.3% 3. 3% 6.6% 38.5% c,s: 1 31.4%-
8.0% 6.4% 26.9% c18:2 16.6%-
54.0% 48.0% -c18:3-
-
-
5.6% -c22:6-
-
-
-
11.6%H H 0 '\".+/ 0 11
-o,,,,
I
'-..+ P· '··.P-O -:;::;N~ / ...:.::;:~~0--a~
0 0 '-..+ 0 0~3
/N'~
ro
0 2 , ,' 3aft
~~o
!a)ra
~ ~
~
\~
- -
~
H H 0 '\".+/ 0 11-o,,,,
I
... + p .,,,, ····P-O ,...N /"''o-
-oV
~o.
o
~N o, H 3 / 'rN
2 H :f OH 0 , , ~rN
OH ( b) )(...
~
~
~
-~
Figure II,ll. Compared to phosphatidyLcholine (a), in sphingomyelin (b) chain following from the formation of a five co-ordinated intermediate, is blocked.
FiguPe II.l2. VePy fast build-up of a five ao-ordinated intermediate in the phosphatidyZsePine headgPoup, due to twofold stabilization.
Phosphatidylinositol possesses a large ligand in the phospho-lipid headgroup, the diameter of which can hardly be changed by the transition from the four to the five co-ordinated intermediate. A possibly formed five co-ordinated intermedi-ate will not be stabilized by intramolecular ring formation so its life-time will not be long enough to induce cluster formation. Phosphatidylethanolamine already possesses a very small headgroup in stretched conformation (see II.1.1.), so intramolecular ring-formation will not be able to influence the angle of tilt of the hydrocarbon chains. Both samples 9 and 10 display a more or less expected rate of ion transport, somewhat slower than that of phosphatidylcholine (sample 1). Addition of these lipids hardly influences the overall lipid fluidity (the ebains display about the same degree of
7
(a)
( b)
Figure II.13. (a) Formation a five co-oPdinated intermediate will not be able to influence hydracaPbon chain tilt in phosphatidylethanolamine.
(b). A five co-ordinated phosphatidylinositol intermediate will not be stabilized by intramolecular ringformation.
