Interactions of mitochondrial structural protein with phospholipids

ARCHIVES

OF

BIOCHEMISTRY

AND

Interactions

BIOPHYSICS

S. H. RICHARDSON; for

Enzyme

(1964)

of Mitochondrial with

Institute

254-260

106,

Structural

Phospholipids’ H. 0. HULTIN3

Research,

Protein

University

Received

AND

of Wisconsin,

August

S. FLEISCHER Madison,

Wisconsin

24, 1963

A new procedure is described for the preparation of structural protein from beef heart mitochondria. Structural protein prepared in this manner combines spontaneously with micellar phospholipids at neutral pH. The interaction is qualitatively and quantitatively similar to the rebinding of phospholipids by “lipid-deficient” mitochondria.

“Structural protein” is the term used to designate the colorless protein first isolated from the mitochondrion by Green and co-workers, which accounts for approximately 60% of the mitochondrial protein (1). At physiological pH it exists as a waterinsoluble polymer; the monomer has a molecular weight of about 23,000 (2). The insoluble character of the protein and the apparent absence of enzymic function suggest that its major role is structural; hence the name. Mitochondria contain 25 % of lipid by weight, more than 90 % of which is phospholipid (3). We have been able to demonstrate that when lipid is extracted from the mito-

chondrion, electron transport activity is lost (4); restoration of activity is dependent on the rebinding of added CoQ and phospholipid (5-8). A requirement for phospholipid has been demonstrated in each of three enzymic complexes which collectively transfer electrons from succinate to oxygen, i.e., succinate -+ CoQ; Co&H, + cytochrome c; reduced cytochrome c -+ O2 (7). Phospholipid, therefore, is not an adventitious component but is an integral and essential part of the enzyme systems of the electron transfer chain. Phospholipids, added in aqueous micellar solution, are rebound spontaneously by the “lipid-deficient”4 mitochondria (5, 6). That rebinding of phospholipid is essential for reactivation of respiratory activity is well established, but the nature of the interaction between the protein and the phospholipid has yet to be characterized. Structural protein prepared as originally described by Green et al. (1) does not combine appreciably with added phospholipid micelles when mixed together at neutral pH. Does this mean that the lipid is not bound to structural protein in situ? Or, alternatively, has the method of preparation so

1 This work was supported in part by the following USPHS grants: Postdoctoral Fellowship grant GDP-16,090 and Graduate Training Grant 5-Tl-GM-88, Division of General Medical Sciences; research grant RG-7674, Division of Research Grants; and research grant HE-00458, National Heart Institute. *Postdoctoral Trainee of the Institute for Enzyme Research, University of Wisconsin, Madision, Wisconsin. Present address: Department of Microbiology, Bowman-Gray School of Medicine, Wake Forest College, Winston-Salem, North Carolina. 3 Postdoctoral Fellow, Division of General Medical Sciences, National Institutes of Health, USPHS. Permanent address: Department of Food Science and Technology, University of Massachusetts, Amherst, Massachusetts.

4 “Lipid-deficient” mitochondria will be used to designate beef heart mitochondria which have been extracted with 10yO water in acetone. This procedure removes the neutral lipids as well as approximately 80% of the phospholipid (4, 5). 254

STRUCTURAL

PROTEIN

AND

altered the structural protein that it can no longer combine with phospholipids under physiological conditions? We assumed the latter situation as a working hypothesis and, accordingly, modified the procedure of Green et al. to eliminate “harsh” steps which might denature the protein. The data reported in this paper shorn that we succeeded in preparing structural protein, which, like “lipid-deficient” mitochondria, is capable of binding phospholipid micelles at neutral pH. EXPERIMENTAL MATERIALS

PROCEDURE AKD

lbTH0~f2

Preparation of structural protein. Structural protein was prepared by two methods. The first method follows the original procedure of Green et al. (1); it involves the use of sodium dodecgl sulfate and bile salts, and the extraction of the protein with methanol at 50”. The second method, to be described, is a modification of the first. 9 suspension of mitochondria in 0.25 M sucrose [Fraction Aa as described by Crane et al. (lo)] is mixed with one-third of its volume of 0.9% KCl; the mixture is homogenized and kept in the frozen state for 15 hours or longer. The thawed homogenate is centrifuged at 78,000g for 20 minutes; the red supernatant fluid, the fluffy layer on top of the packed mitochondria, and the dark brown pellet in the bottom of the tube are all discarded. The mitochondria are resuspended in a volume of 0.9ojc KC1 equal to the original volume and the suspension is centrifuged as before. The washing is repeated until the supernatant fluid becomes a light straw color, the dark brown pellet being removed with each washing. Thorough washing is required to remove the water-soluble proteins from the mitochondria before further fractionation is attempted. The washed mitochondria are suspended at a protein concentration of 20 mg. per milliliter in 0.25 M sucrose and are stored at -20°C. until needed. Sufficient cholate and deoxycholate are added as potassium salts (pH 7.5) to bring their final concentration to 1.0 and 2.0 mg., respectively, per milligram of protein. The addition of the bile salts results in clarification of the mitochondrial suspension. (The use of Duponol has been eliminated from our present procedure.) The resultant brown-green solution is sedimented at 20,OOOg for 20 minutes to remove insoluble material. The hemoproteins in the supernatant solution are reduced by the addition of 2 pg. of sodium dithionite per milligram of protein, and t’he solution is brought’ to 0.12 saturation with a neutral-

PHOSPHOLIPID

INTERACTION

255

ized, saturated solution of ammonium sulfate (4°C.). To ensure a structural protein uncontaminated with heme, the cytochromes must be in the reduced state and the pH must be kept between 7.0 and 7.5 during the addition of ammonium sulfate. After 10 minutes at 4°C. the suspension is centrifuged for 20 minutes at 35,000g. The precipitated protein (essentially white) is homogenized and washed twice in buffer which is 0.05 M in tris-acetate and 0.25 M in sucrose, pH 8. This buffer will be referred to as the “tris-sucrose buffer.” To remove the bile salts and lipids from the protein, a modification of the acetone procedure of Lester and Fleischer (4) is employed. The protein concentration is adjusted to 20 mg. per milliliter with tris-sucrose buffer and is mixed rapidly at O”-3°C. with sufficient amounts of water and acetone to bring the final acetone concentration to 90yG; also, the final volume of the mixture should be 25 times the original volume of the sample. After 3 minutes the major portion of the supernatant fluid is decanted from the readily settling protein; the remaining suspension is homogenized in the cold, and the decanted acetone is then readded. After the protein has been in contact with acetone for a t’otal of 15 minutes, it is sedimented by low-speed centrifugation. The sedimented protein is washed twice with tris-sucrose buffer and is stored at -20°C. in tris-acetate buh’er (0.02 M, pH 7.5) until needed. Structural protein prepared in this manner is utterly insoluble between pH values 2 and 11, and is contaminated with bile salts to the extent of 0.470 as determined in a small-scale preparation in which radioactive bile salts were used. The protein contains 0.2-0.3 pg. of phosphorus per milligram. Since this phosphorus is not extractable by a 2:l mixture of chloroform and methanol, it may not be phospholipid in nature. No heme was demonstrated in the protein by the pyridine hemochromogen procedure of Basford et al. (ll), and the protein showed no absorbancy in the visible region of the spectrum when dissolved in 0.1% Duponol. Nonheme iron was not, detected; nor were sialic acid and similar compounds demonstrated by the Svennerholm (12) technique. It is interesting to note that polymeric structural protein can be “solubilized” at pH 7 by succinylation of 307; of its ninhydrin-titratable groups (13). Mixed mitochondrial phospholipids were prepared from beef heart mitochondria as previously described (5). The composition of the mitochondrial phospholipids has been previously reported by Fleischer et al. (3). 9 preparation of mixed soybean phosphatides (Asolectin) was obtained

256

RICHARDSON,

HULTIN

from Associated Concentrates.5 Cardiolipin was obtained by chromatographing the mixed lipids prepared from beef heart mitochondria (5) on a silicic acid column (5) ; it was also purchased from Sylvana Chemical Co., Orange, New Jersey. Phospholipids were prepared in micellar form as described by Fleischer and Klouwen (6). Protein was determined either by the biuret technique of Gornall et a.1. (14) or by the method of Lowry et al. (15). Total phosphorus was estimated by a modification of the method of Chen et al. (16). The interaction of structural protein with phosphoZipid. The difference in sediment.ability of structural protein suspensions and phospholipid micelles can be used advantageously in evaluating interactions between these two partners. Phospholipid micelles are not sedimented appreciably after 30 minutes at lOO,OOOy, whereas structural protein is readily sedimented in a few minutes in a clinical centrifuge. A suspension of the structural protein essentially devoid of phospholipid (no more than 0.2-0.3 pg. of P per milligram of protein) is mixed with varying amounts of phospholipid in the form of an aqueous micelle; the mixture is incubated at room temperature unless otherwise specified. After an appropriate interval the lipid-protein complex is removed by low-speed centrifugation in the clinical centrifuge; the unbound phospholipid remains in the supernatant fluid. The complex is washed several times with buffer and the ratio of phospholipid to protein is then determined. In some of the experiments structural protein was treated with alkali (pH 12) prior to the addition of phospholipid. The details are given in the legends of the tables. RESULTS

Structural protein, as prepared by the original procedure of Green et al. (l), bound phospholipids in insignificant amounts between pH values 7 and 8 (about 2 pg. of P per milligram of protein) (Tables I and II). This small amount of binding was not 5 The mixed soybean phosphatides were devoid of neutral lipids. The phospholipid composition, analyzed by silicic acid paper chromatography according to the method of Marinetti et al. (17, 18), was lecithin, 317’0 (of total phosphorus); phosphatidylethanolamine, 26%; phosphatidic acid, 11%; cardiolipin, 4y0; phosphatidylinositol (components migrating with the same RI), 18%; two unidentified compounds, 9%; phosphoruscontaining compounds remaining at the origin

wo.

AND

FLEISCHER TABLE

AMOUNT

OF PHOSPHOLIPID

PROTEN

I BOUND

AS A FUNCTION CONCENTRATION

BY STRUCTURAL

OF PHOSPHOIJPID AND OF pH

A suspension of the protein containing 10 mg. of protein per milliliter was mixed with the indicated amount of Asolectin in a buffer which was 0.02 M in tris-acetate and 0.001 M in EDTA, pH 8.0, and the pH was adjusted as indicated. The reaction was allowed to proceed for 10 minutes at room temperature before the pH (where required) was readjusted to 7. The complex was removed, washed, and analyzed as described in Methods. Phospholipid added bP. p/w. protein)

5.2 10.4 15.6 20.8 26.0 a The procedure bile salts methanol

Phospholipid

bound

(~6. P/ak$HpLein)

2.3 2.5 2.4 2.5 2.6

Phospholipid

bound

(pg. P$$pfLin)

5.0 10.5 13.6 18.8 19.8

structural protein was prepared by the of Green et al., in which treatment with and Duponol, and extraction with warm are used (1).

exceeded when the phospholipid concentrations were increased several fold (Table I). It was possible to effect combination of relatively large amounts of the reactants when the structural protein was first treated with alkali (pH 12), and the phospholipid was added at this same pH. Upon neutralization of the reaction mixture the structural protein-phospholipid complex was readily sedimented, washed, and analyzed. The interaction of phospholipids with alkali-treated structural protein was rapid both at 2” and 38” C. (cf. Table II). Essentially the same results were obtained with the mixed phospholipids from mitochondria as with those from soybeans. Chromatographic analysis (17, 18) of the phospholipid moiety of the complex formed by short term interaction (several minutes) in alkali showed that the only phospholipid degraded to any detectable extent by this treatment was cardiolipin. This lipid accounts for about one-fifth of the total lipids of mitochondria. In contrast to structural protein prepared by the original method, structural protein prepared by the method described above combined spontaneously with phospholipids

STRUCTURAL

PROTEIN

AND

PHOSPHOLIPID

TABLE AMOUNT

OF MITOCHONDRIAL

FUNCTION

OF TIME,

Conditions

for interaction

II

PHOSPHOLIPID TEMPERATURE, of structural

257

INTERACTION

BOUND AND

BY

STRUCTURAL

PROTEIN”

DEPOLYMERIZATION

protein

WITH

Time

A. TJntreated structural protein, pH 8, sedimentation at pH 8 B. Structural protein depolymerized at pH 12, interacted with phospholipid at pH 12,* sedimented at pH 7

(min.)

AS A

ALKALI Temperature (“C.)

Phospholipid bound (pg. of P/mg. of protein)

30

25

1.9

1 30 30 30 120

2 2 25 38 2

21.3 22.7 23.1 25.0 25.7

a Prepared according to the method of Green et al. (1). * Structural protein (10 mg. protein per milliliter) was treated with alkali at pH 12 and was incubated for 15 hours at 2” C.; this length of incubation allows for complete depolymerization and ensures reproducibility of the results. The depolymerized structural protein was diluted to 5 mg. protein per milliliter and mixed with 0.5 ml. mixed phospholipid prepared from beef heart mitochondria (300 pg. of phospholipid phosphorus per milliliter of a mixture 0.02 M in tris and 0.001 M in EDTA, pH 8.0). The mixture was then readjusted to pH 12 and incubated at the temperature for the time designated. The pH was then adjusted to 7 by the addition of 0.05 ml. of 1 M tris HCl pH 8.0, followed by the requisite amount of 0.1 il1 HCl. The structural protein-lipid complex was then sedimented in the International Clinical Centrifuge; the residue was washed with 2 ml. of buffer (0.02 M in tris and 0.001 M in EDT,4, pH 8.0) resuspended in buffer, and analyzed. TABLE OF VARIOL-s

&R-DING

PHOSPHOLIPID~

MITOCHON~RIA

of the

treated

AS A FWCTION

PROTEINS AMOUNT

AND

Structural added protein)

protein Phospholipid bound (fig. P/mg. protein)

Mised

mitochondrial phospholipids 15 29 59 Cardiolipin

Mixed 6 .8 8 .6 9 7 7.8 20.1 31.6

7.1

19 38 Asolect.in S 20 40 160

7.3 9.6 10.7 11.9

a The

structural

‘2 IIata

from

per

protein was prepared Fleischer et al. (5). The milligram of protein.

TO ACETONE-EXTRACTED

OF PHOSPHOLIPID

protein

Phospholipid (pg. P/ml.

phorus)

OF THE

in a buffer t.hat. was 0.02 fW in tris-acetate and at room temperature with the indicated amounts of phospholipids reaction mixtures was 2 ml. Aft’er 20 minutes at room temperature as described in illethods.

Structural

mixed

III

TO HTFKCTT:RAL

ADDED

0.001 M in EDTA, pH 8.0, was in the same buffer. The volume the complex was collected and

Acetone

extracted

Phospholipid be. P/mg.

added protein)

mitochondriab Phospholipid bound (fig. P/mg. protein)

mitochondrial phospholipids

22 31 69 Cardiolipin 3.3 6.7 13 26

7.0 9.2 10.3 5.8 7.4 9.8 11.9

-

by the procedure described in this paper. preparation had a residual cont’ent of 3.9 pg. of P (bound

-

phos-

2.58

RICHARDSON, TABLE

EFFECT OF VARIATION TION ON THE AMOUNT TO

HULTIN

IV

IN CONDITIONS OF PHOSPHOLIPID

STRUCTURAL

OF INCUBABOUND

PROTEINS

A solution containing 5 mg. of protein in 1 ml. of buffer (0.02 M in tris-acetate and 0.001 M in EDTA, pH 7.0) was adjusted (if needed) to the indicated pH before additional reagents were added. Asolectin (248 pg. P, i.e., 49.6 pg. P/mg. protein) in the same buffer was added and the pH was readjusted to the specified value. The final volume of the reaction mixture was 2 ml. After incubation for 20 minutes at room temperature the pH was brought to 7.0 where necessary; the complex was sedimented in a clinical centrifuge for 5 minutes and then was washed twice in the same buffer. The phosphorus to protein ratio was determined on the washed pellet. Conditions

Phospholipid (pg. P/mg.

of incubation

10.2 36.3 13.8

pH pH pH

7.0 12.0 12, readjusted to pH 7.0 before addition of phospholipid pH7.0-l 8Murea pH 7.0 + 1 M KC1

= The procedure

structural described

protein in this

bound protein)

16.8 8.1 0.28

(starting material)

was prepared paper.

by

mixture

of

soybean

phospholipids

lipids mine)

(a)

the

zwitterion-type

(lecithin together

of

is

phospho-

constitute

the

greater

part

of

and (b) the acidic phos-

OF ADDED

V BILE

SALTS

BINDING OF ASOLECTIN STRUCTURAL PROTEIN’”

the

and phosphatidylethanola-

the phospholipids;

pholipids account for about one-fifth of the phospholipid. It is therefore not too surprising that the phospholipids from these two sources should be bound in comparable amounts and manner by structural protein (Table III). Cardiolipin, in contrast to mixed phospholipids from either source, is bound quite differently; it is bound more efficiently and in greater quantity. Experiments of the type summarized in Table IV were designed to explore the effect of variations in the conditions of incubation on the extent of binding of phospholipid. The medium in which the reaction was to be carried out was prepared before the reactants were added. At pH 7 appreciable binding of phospholipid to the “native,” as opposed to “denatured,” structural protein was observed, the ratio of phospholipid to protein being similar to that found in the intact mitochondrion (3,s). At pH 12 the amounts of phospholipid that were bound were increased 2.5- to 3.0.fold; but when the protein was first treated with alkali and then neutralized before the phospholipid was added, TABLE

similar in composition to the mixed phospholipids from beef heart mitochondrion in that:

FLEISCHER

EFFECT

in micellar form at and near physiological pH values; it sufficed to mix the phospholipids as aqueous micelles with the structural protein suspended in buffer. The amount of mitochondrial phospholipid bound was concentration-dependent, the binding being more efficient at low than at high concentrations and reaching a maximum of about 12 pg of P per milligram of protein at very high concentrations of phospholipid. Quantitatively, the amount of phospholipid bound by structural protein prepared by the new method was almost equal to that bound by lipid-deficient mitochondria (Table III). The

AND

ON

THE

TO

The structural protein (5 mg. per milliliter) and soybean phospholipids (Asolectin), each in a buffer that was 0.02 M in tris-acetate and 0.001 M in EDTA, pH 7.0, were added separately to buffered solutions containing the amounts of bile salts shown. The volume of the reaction mixture was 1.2 ml. After incubation for 20 minutes at room temperature the complex was removed by centrifugation and treated as described in Methods. Asolectin added (fig. ‘/W.~o-

50 50 50 50 50 50 50 0 The

Bile

salt added

None Deoxycholate Deoxycholate Deoxycholate Cholate Cholate Cholate structural

protein

was

4.2 8.4 12.6 4.2 8.4 12.6

5.2 3.9 2.4 1.8 4.1 4.1 2.8

prepared

by

procedure described in this paper.

the

STRUCTURAL

PROTEIN

ASD

only a small increase was observed. Urea (8 M) in the reaction mixture increased the binding; salt had little effect. Extraction of structural protein with acetone removed essentially all the bile salts. The importance of eliminating bile salts quantitatively is shown in Table V. ,4 series of tubes containing increasing concentrations of bile salts in the diluent was set up; then structural protein and phospholipid were added, and the experiment was conducted as usual. As the bile salt concentration of the medium increased, the ratio of phospholipid to protein in the complex decreased; clearly, the bile salts interfered with complex formation. We have successfully isolated structural protein from the electron transfer particle (ETP) (19) and from the DPN-cytochrome c reductase preparation of Hatefi et al. (20). Also, a protein component with properties similar to those of structural protein has been isolated from DPSH-CoQ reductase (21) and from reduced Co&-cytochrome c reductase (22) during fragmentation of these en zymic complexes into their component hemoproteins and flavoproteins. DISCUSSION

The new procedure for the preparation of structural protein from beef heart mitochondria yields a product which is essentially free from heme, flavin, nonheme iron, sialic acid, bile acid, and lipid. The objective of avoiding protein denaturation dictated the choice of mild reagents and conditions. The efficacy of the new procedure is attested to by the evidence that some of the properties of and the reactions undergone by structural protein prepared by the new procedure are different from those characteristic of structural protein prepared by the original method. More specifically, the data presented in this communication document the spontaneous interaction between undenatured “structural” protein and mitochondrial phospholipids leading to the formation of an insoluble complex. Furthermore, complex formation takes place under conditions simulating the physiological. The reaction has

PHOSPHOLIPID

INTERACTIOY

259

much the same character both qualitatively and quantitatively whether the protein partner is “native” structural protein or “lipid-deficient” mitochondria; also, the characteristics of the interaction are similar, whether the source of the mixed phospholipids is mitochondria or soybeans. When lipids are removed from mitochondria (and submitochondrial particles) by extraction with acetone at low temperamitochondria tures, the “lipid-deficient” retain their ability to rebind phospholipid at physiological pH. Enzymic function (electron transport) is dependent on this recombination with phospholipid. (Phospholipid is readily rebound at neutral pH and electron transport is restored.) (5-8). Similarly, when lipids and bile salts are removed from structural protein by extraction with acetone, the protein retains its capacity to interact with phospholipids under mild conditions. Although structural protein which has been “denatured” in the process of isolation forms insignificantly small amounts of complex in a neutral milieu, it binds relatively large quantities of phospholipids in alkaline media. The “native” structural protein binds significant quantities of phospholipid at pH 7; the amount bound is increased in the presence of 8 M urea and is further increased at pH 12. These observations are interpreted to mean that, as the protein unfolds or is depolymerized, new sites become available for interaction with phospholipids. This binding of excessive amounts of phospholipids by either preparation of structural protein at pH 12 probably has no physiological significance since the amounts bound are far in excess of those applicable to the mitochondrion. However, the extent of binding of phospholipids at pH 7 may well be a measure of the extent of denaturation of a structural protein preparation. “Native” structural protein that is capable of binding phospholipids in neutral solution makes it feasible to embark on model experiments designed to characterize the nature of the protein-lipid interaction. Such experiments will be reported in a future communication (9).

RICHARDSON,

HULTIN

AND

ACKNOWLEDGMENTS We are continued course of Olson and assistance. Buell in gratefully generously Company,

grateful to Dr. David E. Green for his interest and encouragement during the this work. We are indebted to Mrs. Reta Miss Cecilia Stern for expert technical The helpful suggestions of Dr. Mary the preparation of the manuscript are acknowledged. Meat by-products were supplied by Oscar Mayer and Madison, Wisconsin.

10. 11.

12. 13.

REFERENCES 1. GREEN, D. E., TISDALE, H. D., CRIDDLE, R. S., CHEN, P. Y., AND BOCK, R. M., Biothem. Biophys. Res. Commun. 6, 109 (1961). 2. CRIDDLE, R. S., BOCK, R. M., GREEN, D. E., AND TISDALE, H., Biochemzstry 1,827 (1962). 3. FLEISCHER, S., KLOUWEN, H., AND BRIERLEY, G., J. Biol. Chem. 236, 2936 (1963). 4. LESTER, R. L., AND FLEISCHER, S., Biochim. Biophys. Acta 47, 358 (1961). 5. FLEISCHER, S., BRIERLEY, G., KLOUWEN, H., AND SLAUTTERBACK, D. B., J. Biol. Chem. 237, 3264 (1962). 6. FLEISCHER, S., AND KLOUWEN, H., Biochem. Biophys. Res. Commun. 6, 378 (1961). 7. BRIERLEY, G. P., MEROLA, A. J., AND FLEISCHER, S., Biochim. Biophys. Acta 64, 218 (1962). 8. BRIERLEY, G. P., AND MEROLA, A. J., Biochim. Biophys. Acta 64, 205 (1962). 9. FLEISCHEH, S., RICHARDSON, S. H., H~LTIN,

14. 15.

16. 17. 18. 19. 20. 21. 22.

FLEISCHER

H. O., CHAPMAN, A., AND FLEISCHER, B., in preparation. CRANE, F. L., GLENN, J., AND GREEN, D. E., Biochim. Biophys. Acta 22, 475 (1956). BASFORD, R. E., TISDALE, H. D., GLENN, J. L., AND GREEN, D. E., Biochim. Biophys. Acta 24, 107 (1957). SVENNERHOLM, L., Biochim. Biophys. Acta 24, 604 (1957). FRANKEL-CONRAT, H., in “Methods in Enzymology” (S. P. Colowick and H. 0. Kaplan, eds.), Vol. IV, p. 252. Academic Press, New York, 1957. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). LOWRY, O., ROSENBROUGH, 0. H., FARR, N. J., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). CHEN. P. S., TORIBARA, T. Y., AND WARNER, H., Anal. Chem. 28, 1756 (1956). MARINETTI, G. V., ERBLAND, J., AND KOCHEN: J., Federation Proc. 16, 837 (1957). MARINETTI, G. V., AND STOTZ, E., Bi0chi.m. Biophys. Beta 37, 571 (1966). LINNANE, A. W., AND ZIEGLER, D. M., Biochim. Biophys. Acta 29, 630 (1958). HATEFI, Y., HAAVIK, A. G., AND JURTSHUK, P., Biochim. Biophys. Acta 62, 106 (1961). HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E., J. Biol. Chem. 237, 1676 (1962). HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E., J. BioZ. Chem. 237, 1681 (1962).