A fluorescent label for the outer components of the plasma membrane

390

BIOCHIMICA ET BIOPI-fYSICA ACTA

BBA 45069

A FLUORESCENT LABEL FOR THE OUTER COMPONENTS OF T H E PLASMA M E M B R A N E A. H. M A D D Y

Chemical Biology Unil, Department of Zoology, Edinburgh (Great Britain) (l~eceived F e b r u a r y 18th, 1964)

SUMMARY

I. The synthesis of a new fluorescent reagent (4-acetamido,4'-isothiocyanostilbene2,e'-disulphonic acid) for the specific labelling of the outer components of the plasma membrane is described. e. The reaction of the reagent with ox erythrocytes is examined. It is concluded that the reagent reacts with a fixed number of sites on the surface of the cell. 3. The problems of designing reagents of this type, and the nature of the reaction between the stilbene isothiocyanate and erythrocytes are discussed.

INTRODUCTION

The research of recent years has indicated that the plasma membrane of the cell is to be regarded as an active metabolic entity rather than as an inert boundary system. The investigation of cell permeability and other cell-surface phenomena has resulted in manv theories which postulate the presence of regions of special activity in the membrane. These catalytic sites have been considered as specific permeability "carriers"l, '2 and as regions of molecular anisotropy a& The theory of the effects of anisotropy and the precise position of molecules on their role in cellular metabolism (vectorial metabolism) has been developed by MITCHELL6,7 for the plasma membrane and the mitochondrion. The verification of any of these theories depends on a knowledge of the precise molecular architecture of the membrane and a simple analysis of the overall composition adds little to the understanding of the organisation of molecules within the membrane. Furthermore some functionally important components which might be transiently located in the membrane, playing their part in its metabolism and then moving away, m a y well be lost during the isolation of the membrane. Insight into the organisation of the membrane relative to the osmotic barrier can be obtained when enzymic analysis is accompanied by the use of enzymic inhibitors judiciously chosen on the basis of the permeability characteristics of the cell s. As an adjunct to this approach MITCHELLsuggested that a reagent could possibly be designed to react with an intact cell without permeating or damaging it, and so label only those components Abbreviation: SITS, 4-acetamido, 4'-isothiocyanostilbene-2,z'-disulphonic acid. Biochim. Biophvs. Acta, 88 (1964) 39o-399

FLUORESCENT LABELLING OF THE PLASMA MEMBRANE

391

accessible from outside the permeability barrier. Subsequent fraetionation of the cell would reveal those reactive components available at the time of the reaction. The design and synthesis of a fluorescent reagent with these properties (SITS) and its reaction with ox erythrocytes is reported here. Erythrocytes have been used as a model system for the development of the reagent since not only are their plasma membranes readily obtainable in large quantities in a pure state, but the cells are also highly suitable for permeability measurements, and, as damage to the membrane results in the liberation of the highly coloured haemoglobin, serve as good indicators of any membrane destruction by the reagent. The methods developed can now be applied to other dispersable cells. EXPERIMENTAL

The synthesis of 4-acetamido,4'-uitrostilbene-2,2'-disulphonic

acid

IO g of 4-amino,4'-nitrostilbene-2,2'-disulphonic acid (monosodium salt) are suspended in 50 ml of a I : I (v/v) mixture of pyridine and acetic anhydride, and 2 ml of water added. The mixture is heated to 8o °, when the solid dissolves, and kept at this temperature for 3 ° rain. The solution is cooled and the product precipitated by the addition of IO vol. of acetone. This precipitate contains I molecule of pyridine which can be removed b y recrystallization from 5 % NaC1. The product is finally purified b y boiling with charcoal and repeating the recrystallization. Yield of disodium salt : IO g.

The synthesis of 4-acetamido,4'-aminostilbene-2,2'-disulphonic

acid

5 g of 4-acetamido,4'-nitrostilbene-2,2'-disulphonic acid (disodium salt) are dissolved in 50 ml of water and I2.5 g of Na~S in 25 ml of water added. The p H is adjusted to between 3 and 4 b y the dropwise addition of concentrated HC1. The reduction is continued for 30 rain at 80 °. The precipitated sulphur is filtered off and the pale yellow solution made faintly alkaline b y the addition of 4 N NaOH, then boiled to concentrate, and allowed to crystallize. The pale yellow amine is purified with charcoal and recrystallized from water-ethanol. Yield of disodium salt: 3.0 g.

The synthesis of S I T S Cf. Fig. I. 1.5 g of the parent amine are dissolved in 8 ml of I °..o NaC1 and 0. 5 ml thiophosgene added with vigorous shaking. In a few minutes a dense precipitate forms and after 1.5 h the excess thiophosgene is removed by three 3-ml washes of ether. SOaH

SOaH

Fig. I. 4 - A c e t a m i d o , 4 ' - i s o t h i o c y a n o s t i l b e n e - 2 , 2 ' - d i s u l p h o n i c acid.

The ether phase is separated on each occasion by centrifugation. The insoluble isothiocyanate is filtered off and rapidly recrystallized from 1 % NaC1. During recrystallization the liquor is very briefly (3o sec) boiled with charcoal. The pale yellow product is dried over P205. In view of the toxicity of the thiophosgene the whole operation is carried out in a fume cupboard. Yield of disodium salt: i.o g. (Found: C, 37.1 ; H, 3.5 ; N, 5.2; S, 17. 4 %. Calc. for C17Hl~N2OTSaNa 2" 3H20 (the presence of water of crystallization is also indicated b y the infrared spectrum)" C, 37.o; H, 3.3 ; N, 5.I" S, 17. 4 %.) Biochirn. Biophys. Acta, 88 (1964) 3 9 0 - 3 9 9

392

A. tt. MADDY

The measurement of stilbene uptake by ox erythrocytes Fresh ox blood is collected into a c i d - c i t r a t e - d e x t r o s e 9 and washed 5 times with isotonic saline to remove serum and white cells. The prolonged washing is necessary as the serum proteins are only slowly removed: even after 5 washes a trace of protein is found in the next wash. 6 ml of the washed blood are measured into a graduated tube and centrifuged at 2200 x g for 5 rain. The volume of the cell pad is noted (it has a mean value of 2.5 ml), 3 ml of the supernatant removed, and replaced by 3 ml of stilbene solution in buffered isotonic saline (9 vol. 0. 9 % N a C I - I vol. 0.2 M Na2HPO 4KH~PO4 buffer, pH 7.4). The cells are suspended by gentle agitation and after Io rain at room temperature spun down. During the reaction there is a very slight release of haemoglobin from the cells which imparts a pale straw eolour to the supernatant. 3 ml of the supernatant is removed, I ml of 20 % trichloroacetic acid added to precipitate protein, the precipitate spun down and the supernatant diluted to a concentration suitable for the estimation of the stilbene. The estimation is complicated by the lightcatalysed cis-trans isomerisation. In the case of the isothiocyanate the change is detected by a fall in the absorption peak at 335 m/~ with a concomitant increase of the peak at 265 m/~ (see ref. IO). The absorption in the trough at 295 m/~ does not change during several hours in the light. Freshly prepared solutions contain very little of the cis isomer. The test solutions are carefully shielded from bright light, their absorptions measured at 335 m/~ and 295 m/~ and compared with control solutions which have been diluted in the same way but not added to the blood and kept in the same test-tube rack as the experimental tubes throughout the experiment. The value for the amount of stilbene removed by the blood is the same when calculated from measurements at either wavelength. A comparison of the ratio of the absorption at the two wavelengths in a freshly made up solution with that of the solutions used in the above experiment shows that by careful protection from light the absorption at 335 mt~ does not fall by more than 5 % during the experiment. The bisacetamidostilbene has maxima at 325 m/~ and 255 m/x with a stable trough at 285 m/~. In order to calculate the amount of stilbene taken up by the cells the extracellular space of the pad must be known. This is determined by centrifuging cells under the conditions used above in an isotonic saline solution containing sucrose. The sucrose is estimated by hydrolysis in I N HC1 and measurement of the reducing sugars n. The cells occupy 80 % of the pad volume. The effect of time on the uptake is studied by varying the time for which the erythrocytes are suspended in the stilbene solution prior to eentrifugation.

The preparation of labelled ghosts Freshly collected blood is washed five times and incubated for IO rain at room temperature (20 °) in a IOO /,M solution of SITS in isotonic saline buffered with phosphate at pH 7-4. All further operations are carried out at 4 °. Unreacted stilbene is removed by washing the cells six times with isotonic saline. The last two washes have only the faintest trace of fluorescence. The cells are haemolysed overnight in 6 vol. of water. The ghosts are collected by centrifugation at 20000 x g for 20 rain and washed in water until the washings are almost colourless. The pad remains quite red until the remaining haemoglobin is removed by one wash in o.I M phosphate buffer at pH 8.0 and a second wash in o.oi M buffer 9,12, ~3. As haemoglobin which quenches the fluorescence of the stilbene is removed, the fluorescence of the ghosts becomes ap-

Biochim. Biophys. Acla, 88 (1964) 39o-399

FLUORESCENT

LABELLING

OF THE

PLASMA MEMBRANE

393

parent until finally the pale flesh coloured pad is brightly fluorescent under ultraviolet light. The product is stored at --20 ° at p H 7-4 after division into suitable aliquots. I ml of pad possesses a dry weight of 13 mg.

The preparation of globin As haem quenches the fluorescence of stilbene, the detection of any which might have labelled haemoglobin required the preparation of haem-free globin. This was carried out b y the method of MIRSKY AND ANSON14.

RESULTS

AND

DISCUSSION

The design and synthesis of the reagent The essential properties of a reagent which can be used to label groups on only one side of the plasma membrane are: (i) The reagent must not pass through the osmotic barrier of the membrane. A molecule can be made impermeable b y the introduction of sufficient hydrophilic residues to hinder its passage through the predominantly lipoidal plasma membrane. It is desirable to confer hydrophilia on the reagent by an uncharged group such as a sugar residue, but this ideal has not yet proved technically possible, and lipid insolubility has been achieved b y the introduction of sulphonic acid residues. (ii) The reagent must react with the membrane under physiological conditions of temperature, p H and tonicity without disrupting the cell. A number of reagents well known to protein chemistry can react under these conditions, e.g. diazonium salts, acid chlorides, isocyanates and isothiocyanates. In the present investigation an isothiocyanate has been used. (iii) The label must be detectable in small amounts. While a radioactively labelled compound is preferable for quantitative investigations, fluorescence has the advantage of being more easily followed, both during chemical fractionation, and microscopically in the living cell. The well known fluorochromes of antibody studies, e.g. fluorescein, rhodamine B2oo (see refs. 15-17) are large non-polar molecules when in the reactive state, and would have to be heavily substituted with polar residues to render them impermeable. The resulting molecules would resemble histological stains, tending to be strongly adsorbed on to proteins. Any adsorbed molecules would be difficult to distinguish from those covalently bound, and during the fractionation of the reacted membranes the adsorbed dye could exchange with other proteins which were originally unlabelled. As the reagent must be reactive towards protein, a suitable fluorophore must preferably not possess free amino and phenolic groups. Unfortunately these are the very groups which enhance fluorescence 18,19. Any two of these characteristics can be incorporated into a wide range of molecules, but the addition of the third into a molecule of reasonably low molecular weight usually interferes with one of the other two, e.g. the reactivity of diazonium salts is suitable, but the azo dye resulting from the reaction with the protein often quenches the fluorescence present in the unconjugated reagent. Likewise, the reactivity of acid chlorides is satisfactory, but only in a few special cases is it possible to make an acid chloride bearing sulphonate residues. Isothiocyanates are the most suitable reagents Biochim. Biophys. Acta, 88 (1964) 3 9 0 - 3 9 9

394

A.H. MADDY

of those investigated, being preferred to the corresponding isocyanates on account of their greater stability in aqueous solution. The synthesis of SITS, a compound which meets the above three requirements, is described in the experimental section. The fluorescence of the stilbene nucleus has the advantage of not being dependent on the presence of any substituting groups, e.g. amino moieties, and is not quenched by sulphonation. The disadvantages are: (i) it exists in cis and ~raus forms, the cis isomer having a low or zero fluorescence"°; (ii) the light emission is pale blue in colour and could therefore, unless suitable controls are used, l:e confused with that from tissue autofluorescence. (The erythrocyte membrane has no detectable autofluorescence.) An emission at a longer wavelength would be preferable and the search for such a fluorophore that also meets the other requirements listed above is being pursued. The compounds whose synthesis is described in the experimental section are

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Biochim. Biophys. Acta,

- --,

theoretical

88 (T9(:,4) 390 399

FLUORESCENT LABELLING OF THE PLASMA MEMBRANE

395

identified by chromatography and spectroscopy. The starting compound 4-amino,4'nitrostilbene-2,2'-disulphonic acid cannot be directly converted into a suitable reagent by the formation of its isothiocyanate because the nitro group quenches fluorescence. The acetylation of the 4-amino group is followed chromatographically by the disappearance of the yellow spot which couples with diazonium salts, and its replacement by a new spot which does not. The reduction of the 4'-nitro group is characterized by the appearance on a chromatogram of a fluorescent amino compound and the presence in the infrared spectrum of the product of an amino deformation band at 165o cm -1 and an amino-stretching band at 32oo cm 1. Concomitantly the absorption bands characteristic of nitro compounds at 135o and 152o cm 1 disappear. The last stage in the synthesis produces the isothiocyanate which reacts with amino acids and proteins to form fluorescent derivatives and has a strong absorption band assignable to - N - - C - - S stretching 21 at 215o cm 1. The stilbene derivatives are separated by descending paper chromatography in o.2 N NH4OH. The separation is complicated because the solvent separates cis and trans isomers, the cis moving in front of the trans. Each compound therefore gives rise to two spots, and if the paper is dried in daylight and run in the second dimension in the same solvent both these spots again separate into two.

The uptake of S I T S by ox erylhrocytes It can be seen from Figs. 2a and 3 that the total amount of SITS taken up by the cells remains constant as the concentration of SITS is varied. The uptake has a mean value of 12(±6)" IO 9 moles of SITS per ml of packed cells. The curves have been constructed from experiments performed on different days on different samples of blood.

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Fig. 3. The effect of SITS c o n c e n t r a t i o n on t h e n u m b e r of b i n d i n g sites pe r e r y t h r o c y t e .

Most of the scatter is attributable to differences between blood taken from different animals, for the experimental values obtained within each sample are more consistent. At low concentrations therefore the results indicate the presence of a fixed number of highly reactive sites on the erythrocyte. Measurement of the reaction with different quantities of cells shows that the uptake varies linearly with cell number. The analysis of the time course of the reaction reveals that the cells are saturated after less than 5 min in the stilbene solution and that there is no detectable uptake over the ne,,'t 25 min (Fig. 4). Biochim. Biophys. Acta, 88 (1964) 390-399

396

A.H. MADDY

These data indicate that the reaction is confined to sites outside the permeability barrier. An alternative explanation, that the uptake is completed by the rapid attainment of equilibrium concentrations of reagent inside and outside the cells is not tenable since the amount taken up remains constant at much higher stilbene concentrations. The smallness of the quantity (it is equivalent to only I stilbene molecule per 4oo molecules of haemoglobin) itself suggests that the whole of the cell contents is not available to the reagent. Further evidence for the impermeability of the membrane to SITS can be obtained by preparing the globin from the haemoglobin of reacted cells.

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Both the globin prepared from the haemolysate, and from the haemoglobin liberated from the ghosts by buffer at pH 8.o, are free from fluorescence. Thus material accessible from outside the cell becomes labelled while that on the inside (assuming that all the haemoglobin is inside) does not. Such an effect could be caused either by the impermeability of the membrane to the reagent, or by the fact that although the SITS does permeate, insufficient enters to confer any detectable fluorescence on the large quantity of haemoglobin in the cell. It is probable that insufficient stilbene reacts with the cells to confer any detectable fluorescence on the bulk of the haemoglobin, but there is some evidence 9, ~2 for the haemoglobin liberated at pH 8.o being a discrete fraction of the total haemoglobin, and part of the membrane. The uptake of SITS can be shown to be due to covalent binding rather than adsorption by comparing it with the uptake of the non-reactive analogue 4,4'-bis(acetamido)stilbene-2,2'-disnlphonic acid which, although non-reactive would be expected to be adsorbed in the same way as the isothiocyanate. The mean uptake of the bis(acetamido)compound is zero over the whole concentration range studied (Fig. 2b) and remains at zero for at least 3o min (cf. Fig. 4). When erythrocytes are treated with this compound and the ghosts prepared in a manner identical to that used for the SITS ghosts, they show no fluorescence. The fluorescence of the SITS ghosts cannot be washed away. As reaction of the cells with SITS has no measurable effect on the subsequent uptake of the bis(acetamido) analogue it may be concluded that the isothiocyanate treatment does not affect the permeability of the cell towards stilbene disulphonic acids. Biochim. Biophys. Acta, 88 (1964) 390-399

F L U O R E S C E N T L A B E L L I N G OF T H E

PLASMA M E M B R A N E

397

The nature of the reaction

Isothiocyanates are known to react under mild aqueous conditions 23-28 with amino, sulphydryl, tyrosyl, histidyl and sometimes guanidyl groups. The labelling of the cell probably depends on the reaction through one of these groups, probably as a protein side chain, but possibly as part of a lipid or amino sugar 29. The sulphydryl and tyrosyl derivatives are known to be rapidly hydrolysed at pH I I (ref. 30) and as the SITS is not removed from the cell surface at this pH it is reasonable to suppose that it has substituted an amino, histidyl or guanidyl group. This interpretation is not at variance with present concepts of the surface. The cell surface is regarded 31,32 to be predominantly proteinaceous but bearing a significant carbohydrate component which is important antigenically and contains the sialic acid residues responsible for the negative charge on the membrane. BANGHAMet al, aa were unable to find any cationic groups in the surface of shear of human erythrocytes. HEARD ANDSEAMAN34 tosylated red cells in an attempt to discover whether the negative charge on the surface was a resultant of positive and negative groups or entirely due to negative groups and found that tosylation, which was expected to block any amino groups present did not alter the charge significantly. The absence of cationic groups was also indicated by chargereversal experiments. However, these workers did not claim to be able to detect basic groups at a density below 5 % of the acidic groups. In the present study the cells derived from 5 ml of blood (I ml of the 6 ml used in the uptake experiments is accounted for by acid-citrate-dextrose) take up 3o" lO.9 moles of SITS, which taking the number of cells per ml as 8" lO9 (ref. 35) is equivalent to an uptake of 4.5" lO5 molecules per cell (Fig. 3). EYLAR et al. a6 estimated the number of negative charges oi1 the ox erythrocyte surface to be 6.1-IOa and a value of lO.9" lO6 may be calculated from the charge-density figure quoted by SEA51AX AND UI~LE~'BRUCKaT. The number of basic groups required to account for the uptake of SITS is therefore at the limit of the number detectable by electrophoresis even if all the basic groups are located within the surface of shear. Groups may be present in the cell surface outside the permeability barrier yet be inside the surface of shear. BANGHAMet c~l. 3a considered that only those groups within IO .~ of the boundary of shear have an effect on the electrophoretic properties of the cell. A number of lines of evidence indicate the presence of basic groups near the cell surface. STEIN~s has concluded from an analysis of copper binding that the active centre of the glycerol-transport system of human erythrocytes, a site which must be available from outside the permeability barrier, involves N-terminal histidine. Other unidentified N-terminal amino acids were detected by phenylisothiocyanate treatment of ghosts. The blood-group substances contain lysine, arginine, together with gluco~amine and galactosamine ~9. Trypsin removes 25 % of the surface charge of human red cells, and since the enzyme acts upon peptide bonds in the vicinity of a positive charge, it is reasonable to assume that such charges are present at the cell surface. The substitution of the surface of erythrocytes by fluorodinitrobenzene has been reported by FEHER AND MATKOVlCS~° who claim that as no dinitrophenylvaline (the N-terminal acid of haemoglobin) can be detected in their preparations the reaction is confined to the cell surface. It would be surprising if as non-polar a molecule as fluorGdinitrobenzene does not pass through the cell membrane, and the absence of dinitrophenylvaline could well be due to the non-reactivity of the valine residues within the cell under the conditions used. Biochim. Bioph),s..4cta, 88 (I964) 390 3 9 9

398

A. tI. MADDY

The labelling of the ox erythrocyte with SITS has made possible the analysis of the supramolecular organisation of the plasma membrane by this approach. The sites which react with SITS must be isolated and identified. Lipid extraction of the ghosts with chloroform-methanol removes no fluorescence and all attempts to solubilise the fluorescence (variation of salt concentration and pH) have failed with the exception of a small fraction soluble in saturated urea. The proteolipids prepared from the membrane by the method of FOLCH AND LEES4~ show no fluorescence. The stilbene appears to be associated with the protein moiety of the insoluble lipoprotein described by THOMAS and his collaborators 4",% or perhaps the structural protein recently isolated from bovine erythrocytes 44. Possible methods of solubilising this complex are now being explored. ACKNOWLEDGEMENTS

The author is indebted to Dr. P. D. MITCHELL for suggesting this approach to the plasma membrane and for his advice and encouragement, to Dr. D. LEAVER for the infrared spectra and to the Nuffield Foundation for financial support of the Chemical Biology Unit. REFERENCES 1 p. G. LEFEVRE, J. Cellular Comp. Physiol., 53 (1959) I63. T. ROSENBERG AND V~r. WILBRANT, J. Theoret. Biol., 5 (1963) 288. u p. D. MITCHELL, Nature, 18o (1957) I34. 4 p. D. MITCHELL, Bigchem. Soc. Syrup. Cambridge, Engl., 22 (1963) I42. 5 p. D. MITCHELL AND J. M. ~OYLE, Nature, 182 (1958) 327. P. D. MITCHELL in T. \V. GOOOWIN AND O. L1NOBERG, Biological Structure and Functio~z, Vol. 2, A c a d e m i c Press, N e w Y o r k , I 9 6 I , p. 581. 7 p. D. MITCHELL, J. Gem Microbiol., 29 0 9 6 2 ) 25. s p. D. MITCHELL, Biochem. Soc. Symp. Cambridge, Engl., 16 (1959) 73H. M. ANDERSON .aND J. C. TURNER, J. Clin. Invest., 39 (1959) I. 10 F. D E L o s , DETAR AND L. A. CARPINO, J. Am. Chem. Soc., 78 (1956) 475. 11 M. SOMOYGI, J. Biol. Chem., 195 (1952 ) I9. 12 R. L. POST, C. R. MERRITT, C. R. I'[INSOLVlNG AND C. D. ALBRIGHT, J. Biol. Chem., 235 (1961) 1796. la j . T. DODGE, C. MITCHELL ANn D. J. HANAHAN, Arch. Biochem. Biophys., i o o 0 9 6 3 ) I I9. 1~ A. E . MIRSKY AND M. L. ANSON, J . Gen. Physiol., 13 (193 o) 469 . la A . H . COONS, i n J. F. DANIELLI, General Cylochemical 3/Iethods~ Vol. I, A c a d e m i c P r e s s , N e w Y o r k , 1958 , P. 399. 16 H. VAN UEHLEKE, Z. Naturforsch., 13B (1958) 722. iv C. S. CHADWICK AND t,[. C. NAIRN, f . Immunol., 3 (196o) 363 . is M. FURST, H. I~ALLMANN AND F. H. BROWN, J. Chem. Phys., 26 (1957) 1321. 19 R. T. ~VILLIAMS, J. Roy. Inst. Chem., 1959 (1959) 6 I I . 2o G. N. LEWIS, T. T. MAGEL AND D. LIPKIN, J. Am. Chem. Soc., 62 (194 o) 2973. 21 IV. A. MILLER AND W. B. V~;HITE, Z. Electrochem., 64 (196o) 7Ol. 22 F. A. KLIPSTEIN AND H. M. ]{ANNEY, J. Clin. Invest., 39 (196o) 1894. 23 H . FRAENKEL-CONRAT, M. COOPER AND H. S. OLCOTT, jr. Am. Chem. Soc., 67 (1945) 314 . 24 j . FRAENKEL-CONRAT AND H. FRAENKEL-CONRAT, Biochim. Biophys. Acta, 5 ( I 9 5 o) 89. ~5 A. J. FARNWORTH, Biochem. J., 59 (1955) 529. 26 Ix[. HALLAS-MOLLER, Arch. Pharm. Chemi, 52 (1945) 627. z7 j . HOPKINS AND A. ~,VORMAL, Biochem. J., 27 (1933) 74 o. ~8 p. DESNUELLE AND M. ROVERY, Biochim. Biophys. Acla, I (1947) 497. 29 F. D. COLLIXS AND L. XV. V~HEELOON, Biochem. J., 7° (1958) 46. 30 O. L. MILLER, J. Biol. Chem., 146 (1942) 34511 E. PONDER, in J. BRACHET AND A. E. MIRSKY, The Cell, Vol. 2, A c a d e m i c P r e s s , N e w Y o r k , 196I, p. I. a2 L. WEISS, Biochem. Soc. Symp. Cambridge, Engl., 22 (1963) 32. aa A. D. ]3ANGHAM, B. A. PETHICA AND G. V. F. SEAMAN, Biochem. J., 69 (1958) 12.

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J. Gen. Physiol., 44 (t96o) 251. 36 W, S. SPECTOR, Handbook of Biological Data, Saunde rs , W a s h i n g t o n , 1950, p. 275. 36 E . I-I. EYLAR, M. _A_.MADOFF, O.V. •RODY AND H. L. ONCLEY, J. Biol. Chem., 237 (1962) I992. 37 Cv. V. F. SEAMAN AND G. UHLEnBRUCK, Arch. Biochem, Biophys., IOO (1963) 4933s W. D. STEIN, Nature, 181 (1958) 1662. 39 %T T. J. MORGAN, Proc. Roy. Soc. London, Ser. B, 151 (196o) 3o8. i0 T. FEHER AND B. MATKOVlCS, Nature, 187 (196o) 7o2. 41 j . FOLCH AND M. LEES, J. Biol. Chem., 191 (1957) 8o7. a2 E. LEVlNE AND L. E. THOMAS, Exptl. Cell ices., 22 (1961) 363 . as L. E. THOMAS AND E. LEVINE, Exptl. CellRes., 28 (1962) 365 . 44 S. H. RICHARDSOn, H. O. HULTIN AND D. E. GREEN, Proc. Nall. Acad. Sci. U.S., 50 (1963) 821. 34 D . H . I-IEARD AND G. V. F. SEAMAN,

Biochim. Biophys. Acta, 88 (1964) 390-399