Squalene Properties, Lipid-Binding, Function in ...

Proc. Nat. Acad. Sci. USA Vol. 70, No. 1, pp. 265-269, January 1973

Squalene and Sterol Carrier Protein: Structural Properties, Lipid-Binding,

and Function in Cholesterol Biosynthesis

(liver/microsomal enzymes/phospholipids/fatty acids)

MARY C. RITTER AND MARY E. DEMPSEY*

Department of Biochemistry, University of Minnesota, Minneapolis, Minn. 55455 Communicated by William D. McElroy, October 81, 1972

ABSTRACT Squalene and sterol carrier protein of liver plays a general role as a vehicle for cholesterol and its water-insoluble precursors; the carrier protein is essential for enzymic cholesterol synthesis. Liver microsomal enzymes contain a small amount of endogenous carrier protein, which is readily removed by washing or purification of the enzyme. Enzymic conversion to products of a cholesterol precursor carrier protein complex is markedly faster than that for initially unbound sterol. The protomer form of the carrier protein has a molecular weight of 16,000; during sodium dodecyl sulfate gel electrophoresis one band is observed. Phospholipid facilitates the aggregation of the protomer to the oligomer form (>150,000 daltons; purified 720-fold) accompanied by the binding of cholesterol precursors to the oligomer. The carrier protein binds fatty acids as well as cholesterol precursors, suggesting that it may more generally be a lipid carrier protein with "squalene and sterol carrier protein" describing the functional aspects of the lipid carrier in cholesterol biosynthesis. Studies with several steroids and related compounds revealed that the binding sites of lipid carrier protein must contain highly specific hydrophobic and polar regions.

Earlier we reported that squalene and sterol carrier protein (SCP), a heat-stable protein found in the soluble fraction of liver homogenates, is essential for optimum conversion of

water-insoluble precursors to cholesterol by microsomal enzymes (1-8). Aspects of these findings were confirmed by other laboratories, (e.g., refs. 9 and 10). In the two previous articles of this series (5, 6), we also presented evidence that: (i) SCP specifically binds squalene and sterol intermediates, (ii) a noncovalent squalene or sterol* SCP complex (oligomer, >150,000 daltons) appears to form part of the active site of each microsomal enzyme in cholesterol biosynthesis, and (iii) an SCP-like molecule is a component of the high-density lipoprotein fraction of serum. More recently we demonstrated the occurrence of an SCP-like molecule in adrenal preparations catalyzing the initial steps of cholesterol metabolism to steroid hormones (11). Accordingly, we proposed that SCP functions uniquely as a vehicle for water-insoluble precursors during cholesterol biosynthesis and that SCP or a structurally similar protein participates in the transport and metabolism of cholesterol (5, 11).

In this communication we describe the absolute requirement for SCP by microsomal enzymes, effects on conversion

Addreviations: SCP, squalene and sterolcarrier protein; SDS,

sodium dodecyl sulfate.

* To whom requests for reprints should be addressed.

This paper is no. III of a series. The previous papers are refs. 5 and 6.

rates of the interaction of a sterol- SCP complex with microsomal enzymes, properties of the protomer (16,000 daltons) of SCP, optimum conditions for purification of SCP by aggregation of its protomer in the presence of lipid, binding of biologically important lipids other than sterols to SCP, and structural features of the lipid-binding site(s) of SCP. Our evidence suggests SCP should more generally be named a lipid carrier protein with "squalene and sterol carrier protein" describing the functional aspects of lipid carrier protein in sterol biosynthesis, metabolism, and transport.

METHODS AND MATERIALS

Unpurified SCP (i.e., the soluble or upper half of the 105,000 X g supernatant fractions, previously designated Fraction A), Fraction C (microsomes washed once), and Fraction D (Fraction C, purified by solubilization, salt fractionation, and gel filtration) were prepared from rat-liver homogenates as detailed elsewhere (4). Complexes of sterols and other lipids with the oligomer of SCP were made under the conditions of Fig. 1A of ref. 6 and Table 5 of this report. The protomer of SCP was isolated from unpurified SCP by heat treatment

(1000, 5 min) in the presence of 5% (w/v) (NH4)2SO4 and

gel filtration (4-8). SCP of highest purity was obtained by aggregation of protomer-SCP in the presence of sterol and phospholipid, followed by isolation of the oligomer (lipid* SCP complex) with a column of Sephadex G-75 (see conditions of Table 3). Formation of cholesta-5,7-dienol from cholest-7enol by A7-sterol-A5-dehydrogenase (EC 1.3.3.x) was assayed by ultraviolet spectral analysis or the epiperoxide derivative procedure; the amount of cholesterol synthesized from cholesterol precursors was determined by passage of sterols through the dibromide derivative (4). For some experiments (e.g., Table 2) isotopically labeled sterols were separated on silicic acid-Super Cel columns with benzene as eluting agent (12). Organic and phospholipid phosphorus was measured by the method of Bartlett (13). Gel electrophoresis of SCP prepara-

tions was done in 0.1% sodium dodecyl sulfate (SDS) on 10% acrylamide as described by Weber and Osborn (14). Sterol substrates were dissolved in propylene glycol, steroyl esters in dioxane, and fatty acids (sodium salts) in water. Lecithin was microdispersed in 30 mM Tris- HCl buffer (pH 8.5) by

sonication (15). Labeled sterols ([2,4-3H]cholest-7-enol, [4-'4C]cholesta-

5,7-dienol, and [4-14C]cholesteryl-acetate) were synthesized and characterized as described (1-4, 7). [4-'4C]Cholesterol was purchased from Schwarz-Mann Research Laboratories, NAD from Calbiochem, lecithin (egg yolk) from Cyclo Chemi-

265

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266 Biochemistry: Ritter and Dempsey

0

> 1.8

Proc. Nat. Acad. Sci. USA 70 (1973)

I

I

k.00

SDS ELECTROPHORESIS _

0.95 L 0.90 L

Core Peptide of Lysozyme RNase

I.-

mj0.85

0

Pro-SCP

0.80

0.75 1-

Trypsin Inhibitor

1.5 1

1

1

4.0

4.1

4.2

43

4.4

45

LOG MOLECULAR WEIGHT

0.70

I

I

I

4.00 4.08 4.16 4.24 4.32 4.40 LOG MOLECULAR WEIGHT

FIG. 1. Migration during gel filtration (left) and SDS gel electrophoresis (right) of protomer-SCP relative to proteins of known molecular weight. Average electrophoretic mobilities and standard errors were protomer-SCP, 0.86 41 0.04 (10 determinations); trypsin inhibitor, 0.76 4? 0.04 (4 determinations); ribonuclease, 0.93 i 0.02 (4 determinations); core peptide of lysozyme [reduced cyanogen bromide cleavage product, residues 13-105 (16)], 0.97 41 0.01 (3 determinations). The molecular weight of protomer-SCP determined by both techniques was 16,000.

TABLE 1. Removal of endogenous SCP from microsomal

enzyme preparations

A5-Dehydrog-

enase

aacctMi/vimtgyy,

pprrootteeiinn

Activation

by SCP of microsomal

A5-dehy-

Enzyme

SCP +

drogenase

Exp. preparation Washes* enzyme Enzyme (-fold)

it Microsomes

1

8.0 2.0

4.0

Purified

microsomes 1 13.8 0.9

15.3

2t Microsomes

1

8.0 2.0

4.0

2

7.5 1.4

5.4

3

7.2 0.8

9.0

Purified

microsomes 3 17.6 0.0

o

* Preparations were washed with 0.1 M phosphate (pH 7.4) by

centrifugation at 105,000 X g for 60 min.

t Unpurified SCP (16.3 mg of protein, 21.7 mg/ml), when

present, was incubated in 0.1 M phosphate (pH 7.4) with 62 ;&M

cholest-7-enol, 120 nM AY-9944, 1 mM NAD, and 2.2 mg (8.9 mg/ml) of microsomal protein washed once (Fraction C) or 0.9 mg (3.6 mg/ml) of purified microsomal protein (Fraction D) (total incubation volume, 1.2 ml) for 30 min at 370 under oxygen

(4). t Unpurified SCP, when present, was incubated with 2.3 mg

(9.2 mg/ml) of microsomal protein washed once, or 2.2 mg (8.8 mg/ml) of the protein washed twice, or 2.9 mg (11.6 mg/ml) of the protein washed three times, or 0.7 mg (2.8 mg/ml) of purified microsomal protein prepared from microsomes washed three

times; other conditions were as given for Exp. 1.

cal, and crystalline proteins of known molecular weight from Sigma Chemical Co. All other chemicals were reagent or anayltical grade.

RESULTS AND DISCUSSION

Absolute Requirement for SCP by Microsomal Enzymes. In previous reports we showed that conversion of waterinsoluble precursors to cholesterol by microsomal enzymes is stimulated at least 4-fold by SCP, i.e., a small amount of enzymic activity is usually detected in the absence of SCP (1-8) (see also Table 2). It was important to establish whether or not the activity in the absence of SCP was due to incomplete removal of residual SCP during preparation of the enzymes. The results of Table 1 (obtained with a preparation of A7-sterol A5-dehydrogenase) demonstrate that endogenous SCP was gradually removed by repeated washing or by purification of the microsomal enzyme (Exp. 1 and 2), resulting in increased activation by added SCP. Complete removal of residual SCP and infinite activation of Ar5-dehydrogenase by added SCP required repeated washings of the enzyme followed by purification (Exp. 2, Table 1). In some enzyme preparations, residual SCP was not detectable (see dihydrolanosterol, Table 2) or was totally removed by the purification procedure, and extra washings were not required (see Table 4). These findings (Table 1) are typical of those obtained with other microsomal enzymes of cholesterol biosynthesis; they demonstrate the absolute requirement for SCP by these enzymes. Furthermore, similar experiments with purified SCP showed that effects described in Table 1 are specific for SCP and not caused by other components in unpurified SCP.

Effects of Interaction of Sterol * SCP Complex with Microsomal Enzymes. We demonstrated previously that all water-insoluble

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Proc. Nat. Acad. Sci. USA 70 (1973)

Squalene and Sterol Carrier Protein 267

TABLE 2. Increased conversion rate to products of a cholesterol precursor * SCP complex over that of initially unbound cholesterol

precursor and SCP

Cholesterol precursor3

[24,25-3H]Dihydrolanosterol (C-30)

Dihydro * SCP Dihydro + SCP Dihydro; no SCP

[3a-3H]Cholesta-7,24-dienol (C-27) A7,24. SCP A7,24 + SCP

A7,24; no SCP

[2,4-3H] Cholest-7-enol (C-27)

A7*SCP A7+ SCP

A7; no SCP

[4-14C] Cholesta-5,7-dienol (C-27) A6,7*SCP

A6'7 + SCP A6,7; no SCP

Total sterol

synthesized (/AM)

during 30-min incubation

0. 8b 0. lc 0.0

1.6d 0. 7e 0.2e

0. 9f 0.4' 0. 1f

1. 4g

0.5w

0.2g

a Cholesterol precursors were bound to SCP (oligomer form) as given with Table 3 and in ref 6. Incubations (total volume, 1.9 ml) contained 8.4 mg of SCP protein (6.0 mg/ml, when

present), 3.4 mg of microsomal protein (8.5 mg/ml) (Fraction C), and initially unbound or bound precursor: 1 ,LM [24,25-3H]-

dihydrolanosterol; or 2.2 1sM [3a-3H] cholesta-7,24-dienol; or

1 jAM [2,4-3H] cholest-7-enol and 120 nM AY-9944; or 1.0 ,uM [4-14C]cholesta-5,7-dienol. All incubations were done for 30 min. Other conditions were as described (4); see also Table 1 and Methods.

bHydroxylated C-29 and C-28 compounds and cholest-7-enol. Hydroxylated C-29 and C-28 compounds. d Cholesta-5,7,24-trienol, cholest-7-enol, and cholesterol. Cholesta-5,7,24-trienol and cholest-7 enol. f Cholesta-5,7-dienol. g Cholesterol.

precursors of cholesterol are capable of complexing with SCP (5-8). The data of Table 2 (obtained with four representative cholesterol precursors) further show that conversion to products by microsomal enzymes of a sterol present in a sterol * SCP complex (oligomer form of SCP) is markedly faster than conversion of initially unbound sterol. Results

U^

FIG. 2. SDS gel electrophoretic patterns of unpurified SCP (top) and protomer-SCP (bottom).

of similar studies with various other cholesterol precursors established that in all cases the conversion of a precursor * SCP complex to cholesterol or other sterols is increased 2-fold or more over that of initially unbound precursor. In this regard, the absolute dependency on SCP for detectable enzymic activity was just described (e.g., Table 1). The probable explanation for the increased conversion rate to products of a precursor SCP complex is given by data obtained with a cholest-7-enol * SCP complex. The apparent Km for the enzymic conversion of cholest-7-enol SCP to cholesta-5,7-dienol SCP was 10 JAM at a ratio of SCP protein to enzyme protein of 10.2/1 (the optimum ratio for this reaction); at a suboptimal protein ratio (5.1/1), the apparent Km for substrate was 30 JAM. At both ratios of SCP to enzyme protein, the maximum rate of the reaction was not markedly changed. These results indicate that the microsomal enzymes have a high affinity for a preformed sterol * SCP complex (the oligomer) that participates at the active site of each enzyme.

Properties of the Protomer of SCP. Heat treatment at high ionic strength causes dissociation of the oligomer of SCP to a low molecular weight protein (protomer-SCP) and loss of associated sterol (5-8). As indicated in Fig. 1, the apparent molecular weight of protomer-SCP determined by gel filtration and SDS gel electrophoresis is 16,000. Protomer-SCP migrates as a single band during SDS gel electrophoresis; a protein with the same mobility as protomer-SCP is present in unpurified SCP (Fig. 2). During preparation of protomerSCP, we observe small amounts of aggregates (30,000-60,000 daltons) of protomer-SCP (see Fig. 1B of ref. 6). These aggregates do not contain bound sterols and are most probably the SCP preparations obtained in the absence of heat treatment at high ionic strength by Scallen et al. (17).

Spectral analysis of protomer-SCP revealed no measurable absorbance in the visible region (350-500 nm); in the

TABLE 3. Influence of phospholipid on formation of aggregated SCP and binding of cholest-7-enol

Addition

of phospholipid

+

Formation of aggregated SCP*

u~g

% of protomer-SCPt

1335

2.9

5250

11.7

Cholest-7-enol bound to aggregated SCP

nmol

% of initialt

18

17.3

81

77.9

Organic phosphorus

bound to aggregated SCP

uAmol

% of initialt

0.8

5.3

2.3

13.7

* Aggregated SCP (oligomer form) was obtained by incubation in 0.3 M Tris.HCl buffer, pH 7.4, of 45 mg of protomer-SCP (con-

taining 15 ,umol of organic phosphorus) with 104 nmol of [2,4-3H] cholest-7-enol and, when present, 46 jumol of lecithin (equivalent to 1.8

/Amol of organic phosphorus) for 30 min at 370 under nitrogen, followed by separation of the protomer and oligomer on Sephadex G-75 (6)

with the Tris * HCl buffer as eluant.

t Expressed as the percentage of total initial protomer-SCP protein, cholest-7-enol, or organic phosphorus applied to the gel column

that emerged with the oligomer of SCP and lipid.

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268 Biochemistry: Ritter and Dempsey

TABLE 4. Purification of aggregated SCP

Total

SCP

Specific Pun-

SCP protein Total activity$ fication Yield

preparation (mg) activityt (AM/mg) (-fold) (%)

Unpurified

SCP

2100

21

Aggregated

SCP*

1.8 13

0.01

1 100

7.2

720 62

* The oligomer, sterol SCP complex, was formed from protomer-SCP in the presence of lecithin and [2,4-'H]cholest-7-enol

(see conditions of Table 3).

t SCP activation ability was measured by incubation (total volume, 1.04: ml) of 5.5 ,M [2,4-'H] cholest-7-enol, either initially

unbound to 14 mg of unpurified SCP or bound to 200 ,ug of aggregated SCP protein, and 1.3 mg of purified microsomal A5dehydrogenase protein (Fraction D). Other conditions were as

given with Table 1. T Specific activity is expressed as MM [2,4-'H] cholesta-5,7-

dienol synthesized in 30 min per mg of unpurified or aggregated SCP protein; no formation of [2,4-'HI cholesta-5,7-dienol was

detected in the absence of SCP.

ultraviolet region, protomer-SCP has a characteristic A280/ A260 of 0.68. The relatively high absorbance of protomerSCP at 260 nm may be due to nucleotide contamination, e.g., we reported previously that SCP binds pyridine nucleotide as well as sterols (6). Nucleotide contamination may also contribute to the amount of organic phosphorus (100300 nmol/mg protein) present in protomer-SCP preparations (Table 3). A typical unpurified SCP preparation contains 1.2 ,umol of organic phosphorus per mg of protein. The organic phosphorus present in protomer-SCP may also reflect some

bound phospholipid.

Influence of Phospholipid on Aggregation and Purification of SCP. The functional similarities of one of the human plasma lipoprotein peptides and human liver-SCP are now well characterized (6, 18). These findings led us to investigate possible additional similarities between plasma lipoproteins and SCP, in particular the interaction of SCP with phospholipids. During these studies we made the striking observation that phospholipid facilitated aggregation of protomer-SCP and sterol binding. The presence of lecithin resulted in a 4-fold increase in oligomer formation accompanied by a similar increase in bound sterol and organic phosphorus (Table 3). It is probable that some of the organic phosphorus present in the sterol SCP complex formed in the presence of lecithin represents bound lecithin; at present this point has not been rigorously established. The influence of phospholipid on aggregation and sterol binding of protomer-SCP was used to obtain a 720-fold purification of the sterol SCP complex

in 62% yield, based on activation of microsomal A'-dehydro-

genase (Table 4). The latter preparation of SCP may be of ultimate purity, i.e., the sterol- SCP complex elutes in the void volume of a Sephadex G-75 column and is thus widely separated from protomer-SCP (see Fig. 1C of ref. 6).

Binding of Biologically Important Lipids Other Than Sterols to SCP. We reported previously that cholesterol, its waterinsoluble precursors, ,3-sitosterol, and hydroxy-derivatives of cholestane (in particular, 3j3, 5a, 6t3-cholestanetriol) bind

Proc. Nat. Acad. Sci. USA 70 (1973)

to SCP; bile acids, steroid hormones, cholestane, and ketoderivatives of cholestane (not cholesterol precursors) are not bound or poorly bound to SCP (5-8). Additional studies (Table 5) were undertaken to define further structural requirements for lipid-binding to SCP. Esterification of cholesterol and another representative sterol (cholesta-5,7,22trienol) results in markedly decreased sterol-binding to SCP (Table 5). Also, the chain length and saturation of the esterified fatty acid appear to influence the observed amount of

binding. In contrast, free fatty acids longer than six carbon

atoms are bound to SCP regardless of the degree of their saturation.

Structural Features of the Lipid Binding Site(s) of SCP. The binding studies just described and our earlier work demonstrate that SCP not only forms high molecular weight complexes with water-insoluble cholesterol precursors [Table 2 and (6-8)], but also with fatty acids (Table 5) and probably phospholipids (Table 3). These results suggest that a more appropriate name for SCP is lipid carrier protein with "squalene and sterol carrier protein", describing the function of lipid carrier protein in cholesterol biosynthesis, metabolism, and transport.

Regarding structural features of the lipid-binding site(s), it is apparent that SCP (lipid carrier protein) must contain: (i) a hydrophobic region to accommodate the sterol nucleus and its side chain or the long hydrocarbon chains of fatty acids and (ii) a hydrophilic region for the binding of hydroxyl groups and other polar sites (e.g., areas of unsaturation) in the lipid molecules. It is possible that SCP contains a thin cleft in its tertiary structure; the flat steroid molecule would reside within the hydrophobic interior of the cleft with the

TABLE 5. Binding of cholesterol, cholesteroyl esters, fatty acids, and related compounds to aggregated SCP

Compound

Cholesterol, cholesteroyl esters, and related compounds

[4-14C] Cholesterol [4-14C] Cholesteroyl-acetate

[7ca-3H] Cholesteroyl-palmitate

[4-14C] Cholesteroyl-stearate

[ 1_14C] Cholesteroyl-oleate

[4-14C] Cholesta-5,7,22-trienol [4-14C] Cholesta-5,7,22-trienyl-acetate

Fatty acids

[1-14C] Caproic acid (C-6)

[[(11-_1144CC]]MPyarlimsittiicc

acid acid

(C-14) (C-16)

[1-14C]Stearic acid (C-18)

[[11--1144CC]]OLlieniocleaicciadci(dC-(1C8-;18lc;o92)w6)

Binding*

(%)

90 35 10 7 39 90 62

78 100 100 100 100 100

* Binding assays were performed by heat treatment of un-

pfcureranictftriiieofdnugaSwtCiitoPhn,(t1hi0en0c0,ucboa1mtipmoionnu)n,(d3r70e(,m25ov1a5Mulg)m,oifnf)oclolaoogfwueltdahteebdysup(prNoeHtre4ni)an2tSabOny4t

precipitation (40-80% saturation), and finally by gel filtration (Sephadex G-25) (6, 7). Binding is expressed as the percentage of the total radioactivity applied to the gel column that emerged

with the SCP complex (oligomer form).

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Proc. Nat. Acad. Sci. USA 70 (1978)

hydroxyl group(s) at or near the surface of the protein. A cleft is present at the functional centers of myoglobin (19), lysozyme (20), and ribonuclease (21); a cleft may also be present in the recently discovered retinol-binding protein (22). Upon specific interaction of a cholesterol precursor * SCP complex with a microsomal enzyme a conformational change would occur, followed by the catalytic step and transfer of the sterol - SCP complex to the next microsomal enzyme. This hypothesis is in accord with our findings that a sterol present in a sterol SCP complex is more readily available to the catalytic site of a microsomal enzyme (Table 2) and that inhibitors of specific steps in cholesterol biosynthesis block formation of the activated conformation of a sterolSCP * enzyme (complex (e.g., refs. 7 and 23).

We gratefully acknowledge the following gifts: cholesteroyl esters from Dr. R. St. Clair, Bowman-Gray School of Medicine; cholesta-5,7,22-trienol and cholesta-5,7,22-trienyl-acetate from Dr. R. Conner, Bryn Mawr College; fatty acids from Dr. R. Johnson, University of Minnesota and Dr. R. Holman, Hormel Institute; dihydrolanosterol from Dr. E. Grossi Paoletti, University of Milan; and cholesta-7,24-dienol from Dr. I. D. Frantz, Jr., University of Minnesota; core peptide (residues 13-105) of lysozyme from Dr. D. Wetlaufer, University of Minnesota. The work was supported by USPHS Grants HE-8634 and HE-6314 and predoctoral Fellowship (GM42265) to M. C. Ritter.

1. Dempsey, M. E., Seaton, J. D., Schroepfer, G. J., Jr. & Trockman, R. W. (1964) J. Biol. Chem. 239, 1381.

2. Dempsey, M. E. (1965) J. Biol. Chem. 240, 4176. 3. Dempsey, M. E. (1968) Ann. N.Y. Acad. Sci. 148, 631. 4. Dempsey, M. E. (1969) in Methods in Enzymology, ed.

Clayton, R. B. (Academic Press, New York), Vol. XV, pp. 505.

Squalene and Sterol Carrier Protein 269

5. Ritter, M. C. & Dempsey, M. E. (1970) Biochem. Biophys. Res. Commun. 38, 921.

6. Ritter, M. C. & Dempsey, M. E: (1971) J. Biol. Chem. 246, 1536.

7. Ritter, M. C. (1971) Ph.D. Thesis, University of Minnesota. 8. Dempsey, M. E. (1971) in The Chemistry of Brain Develop-

ment, eds. Paoletti, R. & Davison, A. N. (Plenum Press, New York), pp. 31. 9. Scallen, T. J., Schuster, M. E. & Dhar, A. K. (1971) J. Biol. Chem. 246, 224. 10. Rilling, H. C. (1972) Biochem. Biophys. Res. Commun. 46, 470. 11. Kan, K. W., Ritter, M. C., Ungar, F. & Dempsey, M. E. (1972) Biochem. Biophys. Res. Commun. 48, 423. 12. Frantz, I. D., Jr. (1963) J. Lipid Res. 4, 176. 13. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466. 14. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406.

15. Fleischer, S. & Fleisher, B. (1967) in Methods inv Enzymology,

eds. Estabrook, R. W. & Pullman, M. E. (Academic Press, New York), Vol. X, pp. 406. 16. Bonavida, B., Miller, A. & Sercarz, E. E. (1969) Biochemistry 8, 968. 17. Scallen, T. J., Srikantaiah, M. V., Skrdlant, H. B. & Hansbury, E. (1972) Fed. Proc. 31, 429. 18. Dempsey, M. E., Ritter, M. C. & Lux, S. E. (1972) Fed. Proc. 31, 430. 19. Kendrew, J. C. (1963) Science 139, 1259. 20. Blake, C. C. F., Johnson, L. N., Mair, G. A., North, C. T., Phillips, D. C. & Sarma, V. R. (1967) Proc. Roy. Soc. Ser. B. 167, 378.

21. Kartha, G., Bello, J. & Harker, D. (1967) Nature 213, 862.

22. Kanai, M., Raz, A. & Goodman, D. S. (1968) J. Clin.

Invest. 47, 2025. 23. Witiak, D. T., Parker, R. A., Brann, D. R., Dempsey, M.

E., Ritter, M. C., Connor, W. E. & Brahmankar, D. M. (1971) J. Med. Chem. 14, 216.

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