The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria

Biochimica et Biophysica Acta, 791 (1984) 375-383

375

Elsevier BBA 32070

THE DOMAIN STRUCTURE OF T H E CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME P-450 FROM BOVINE ADRENOCORTICAL MITOCHONDRIA LOCALIZATION OF HAEM GROUP AND DOMAINS IN THE POLYPEPTIDE CHAIN V.L. CHASHCHIN, V.1. VASILEVSKY, V.M. SHKUMATOV, V.N. LAPKO, T.B. ADAMOVICH, T.M. BERIKBAEVA and A.A. AKHREM

Laboratory of Protein Chemistry, Institute of Bioorganic Chemistry, B.S.S.R. Academy of Sciences, Zhodinskaya 5, Minsk, 220600 (U.S.S.R.) (Received June 14th, 1984) (Revised manuscript received September llth, 1984)

Key words: Cytochrome P-450; Domain structure; Trypsin digestion; (Bovine adrenal cortex)

Cytochrome P-450~.~ consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450~ in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and 1I to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450~. The pathways of limited trypsinolysis of the native cytochrome P-450~ have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyI-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.

Introduction In our previous publication [1] we have shown that the controlled limited trypsinolysis of cytochrome P-450~ from adrenal cortex mitochondria (P-450s~) reveals its native molecule organization as two domains. The haemoprotein molecule is composed of two globules (domains) concurrent to Abbreviations: P-450w.c, adrenal mitochondrial cytochrome P450 which functions in the cholesterol side-chain cleavage reaction; P-450(I + II), trypsin-modified form of P-450~o~. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

fragments in the polypeptide chain with M r 27 000 and 22 000. The P-450~ modified by trypsin up to the equimolar mixture of domains retains its major spectral and functional properties characteristic of the native protein, which supports the assumption of a rigid noncovalent interaction between the domains [1]. The localization of the haem group in the native protein molecule, which also assumes the localization of a catalytic centre, is an important aspect of the structure-functional organization of cytochromes P-450 from various sources [2-4]. In this work

376

the data on separation of P-450s~ domains, their localization in the polypeptide chain, and the localization of the haem group in one of the domains, are presented as a prerequisite to the study of the primary structure of P-450~c and its interrelation with function. Also reported are the results from the study of the amino acid sequence of sites in the P-450s~c polypeptide chain containing the surface cysteine residues. Materials and Methods

Standard buffer systems. Buffer A, 50 mM sodium phosphate buffer (pH 7.2)/1 M NaCI/ 0.3% sodium cholate/0.3% Tween 80/1 mM EDTA; buffer B, 50 mM sodium phosphate buffer (pH 7.2)/1 M NaC1/0.3% sodium cholate/1 mM EDTA. All the other buffer systems contain 0.1 mM EDTA. The isolation of P-450~, its cleavage with trypsin and isolation of P-450(I + II) were performed as described earlier [1]. Chromatography on Sephadex G-200. The P4500 + II) obtained, 0.5/~mol, was stored at 4°C for 0, 24, 48 and 120 h in buffer A. At the above intervals, aliquots of P-450(I + II), 60 nmol, were withdrawn and subjected to gel-chromatography on a Sephadex G-200 column (1.4 × 31 cm) equilibrated with the same buffer. Column eluates were monitored with the Uvicord S insmmaent adjusted with a two-channel LKB 2210-recorder. Covalent chromatography on activated thiopropyl-Sepharose. A typical experiment on separation of domains involved 1 #mol P-450(I + II) and 1.5 g thiopropyl-Sepharose 6B (Pharmacia, Sweden), equilibrated with buffer B. A mixture of fragments (20 ml) was passed through a column with activated thiopropyl-Sepharose at 20°C, at the rate of 10 m l / h . Under these conditions a covalent immobilization of the trypsin-modified protein to the adsorbent occurs as a result of the reaction of thiol-disulphide exchange among the accessible SH-groups of protein and hydroxypropyl 2-pyridyldisulphide bound with Sepharose. The gel was washed by buffer B up to the disappearance in the eluate of 2-thiopyridone [5] and of unreacted protein. The column was equilibrated with 50 mM phosphate buffer (pH 7.2) and then washed in

sequence with the same buffer containing 7 M guanidine and, finally, by the same solution with 0.1 M 2-mercaptoethanol. Isolation of peptides containing the surface cysteine residues. P-450scc, 0.8 /xmol, was immobilized in a Corex tube on the activated thiopropylSepharose (6 ml) at 25°C for 45 min in buffer B. Then the gel was stored for 20 min on a bath at 95°C, washed with 50 mM phosphate buffer (pH 7.2)/7 M guanidine and equilibrated with 0.2 M ammonium bicarbonate buffer (pH 8.2). The P450s~ immobilized on thiol-Sepharose was hydrolysed by chymotrypsin in an inert-gas atmosphere with continuous stirring at the enzyme/substrate ratio of 1 : 30 (w/w) at 37°C. After 18 h hydrolysis, the Sepharose was successively washed with 0.2 M ammonium bicarbonate buffer (pH 8.2)/50 mM phosphate buffer (pH 7.2)/7 M guanidine, and then again with 0.2 M ammonium bicarbonate buffer (pH 8.2) with 0.1 M 2-mercaptoethanol. The obtained peptide fraction was liophylized and subjected to carboxymethylation as in Ref. 6, and then desalted on Bio-Gel P-2 in 80% formic acid. The peptide fractions were separated on a column (0.8 x 25 cm) of Servachrom Si 100 C8 phase (100 gm) using as eluate a solution with increasing concentration of acetonitrile in 0.1% aqueous trifluoroacetic acid. Analytical procedures. The analytic gel-electrophoresis under denaturation conditions was carfled out following Ref. 7. When detecting any residual haem-peroxidase activity, the samples were treated in 0.1% SDS without 2-mercaptoethanol prior to gel-electrophoresis. The samples of P450(I + II) were stored at 4°C in 0.1% SDS; 1-2 nmol P-450(I + II) were employed for electrophoresis. The electrophoresis and determination of the residual haem-peroxidase activity in the presence of tetramethylbenzidine were performed as in Ref. 8. The amino acid analysis was performed following the method described in Ref. 9. Three parallel amino acid analyses were carried out for each sample on the LKB-3201 (Sweden) and LC 2000 (Biotronics, F.R.G.) amino acid analyzers after 24, 48 and 72 h of acidic digestion. Tryptophan and cysteine were determined by the techniques described in Refs. 10 and 11, respectively. The N-terminal amino acids were determined following

377

the method reported in Ref. 12 as dansyl derivatives. The C-terminal amino acids were determined with carboxypeptidase Y [13]. The amino acid sequence was determined by the method of Ref. 14. The haem was removed from the protein sampies with acidified acetone [15]. Results

Separation of domains I and 11 under nondenaturation conditions The aim of the present section of our work was to seek conditions for the separation of domains under mild conditions providing, on the one hand, an effective separation, and, on the other, maximum retention of the haem group noncovalently bound with the protein. An effective separation of domains I and II requires a preliminary dissociation of the domain complex. The reagents most frequently used for this purpose, SDS, urea and guanidine, proved ineffective, since treatment by them leads to a significant dissociation of noncovalently bound haem, whereas treatment with them in combination with 2-mercaptoethanol is accompanied by a practically complete removal of the haem group. The content of a component with •max ~---423 nm in the difference absorption spectrum of the carbon monoxide complex of P-450(I + II) has

been found to increase as a result of its prolonged incubation (some days, 4°C) in buffer A (see Materials and Methods). In a general case it might be evidence for the disturbance in native coordination of axial ligands of different types of cytochromes P-450. After this preparation of P-450(I + II) was subjected to gel chromatography on precalibrated Sephadex G-200 column, two fractions have resulted with approximate M r values 400000 and 110000, respectively. The analytical gel electropboresis of these two fractions in the presence of SDS showed the fraction with M r 400000 to be enriched with domain II, whereas the other is enriched with domain I. We have assumed that under prolonged storage of P-450(I + II) in the presence of detergents (sodium cholate and Tween 80) and at high ionic strength, a gradual dissociation of a complex of domains I and II occurs. Additionally, the above dissociation of domains seems to be accompanied by the formation of their aggregate forms with different molecular masses, which leads to the appearance under chromatography of fractions with redistributed content of domains. To prove our assumption, we have investigated the behaviour of P-450(I + II) under gel-electrophoresis after its preliminary storage under the above-described conditions for various time intervals. In Fig. 1 the alterations of chromatographic

TABLE I D I S T R I B U T I O N O F H A E M A N D P R O T E I N M A T E R I A L BY F R A C T I O N S A A N D B A F T E R G E L - C H R O M A T O G R A P H Y O F P-450(I + II) U N D E R N O N D E N A T U R A T I O N C O N D I T I O N S Incubation

Fraction A

time before gel-chromatography (h)

Haem yield (%) ~

Fraction B A 41s//12so b

Content of domains I and II (%) ¢

Haem yield (%) a

A 418/A 2so b

Content of domains I and II (%) c I 50 II 50 1 60 II 40 I 70 II 30 I 85 II 15

0

-

-

-

91

0.85

24

9

0.5

87

0.9

48

12

0.35

83

1.08

120

8

0.2

I 25 II 75 1 20 II 80 I 11 II 89

88

1.27

" The absorption units in a sample at 418 n m before gel-chromatography are taken to be 100%. b A 4 1 s / A z S ° index of the starting P-450(I + II) was 0.82.

c The s u m of molar a m o u n t s of I and II, detected after SDS-gel elcctrophorcsis in each fraction (A a n d B) is taken to be 100%.

378

profiles depending on the incubation time of P450(1 + II) are presented. It should be noted that under our experimental conditions the freshly prepared P-450(I + II) was chromatographed as a single peak with M r 110000 (Fig. 1A). The formation of another fraction with M r 400 000 occurred with prolongation of preliminary incubation of P-450(I + II) (Fig. 1B, C, D). Presented in Table I are the data from the study of distribution of the haem into fractions (evaluated from absorption spectra of the material of individual fractions by the A418//A280 ratio) and domains I and II (analytical SDS-gel electrophoresis data). From the data presented, it is seen that the formation of a fraction with M r 400000 at prolonged incubation of P-450(I + II) preparation results from a decrease of domain II content in the fraction with M r 110000. The enrichment of the latter fraction with domain I, whose maximum content amounted to 85% (Table I), was accompanied by the increase in A418/A28o ratio from 0.85 to 1.27. The yield in haem under chromatographic experiments was in all the cases about 90%, including the residual content of haem in fractions with M r 400000. All the above allows us to conclude that the haem is likely to be associated with domain I. Gel-electrophoresis of P-450(I + II) under conditions providing the retention of the residual haem-peroxidase activity with subsequent staining of gels in the presence of tetramethylbenzidine and hydrogen peroxide [8] proved that the haem-associated activity is located in the region of domain I. Thus, it can be claimed that the haem is associated with the globule of domain I.

0.2--

0.1

a2--

0.1 - -

o

(~2

0.1

Q2--

0.1--

I

I

I

I

I

5

10

15

20

25

Elution volume

(ml)

Fig. 1. Gel-chromatography of P-450(I + II) after storage in 50 mM sodium phosphate buffer (pH 7.2)/1 M NaCI/0.3% sodium cholate/0.3% Tween 80/1 mM EDTA for 0 (A), 24 (B), 48 (C) and 120 h (D). /'-450(1 + II) (60 nmol) was applied to a column

Separation of domains I and H under denaturation conditions Due to difficulties with removal of detergent upon the separation of domains under nondenaturation conditions, and because of their partial mutual contamination, the approach mentioned has not been used for the preparation of domains suitable for a structural analysis.

(1.4× 31 cm) of Sephadex G-200 and eluted with the same buffer. Fractions A (M, 400000) and B (M r 110000) shown by solid bars were pooled and analyzed.

379 The application of the preparative SDS-gel electrophoresis, gel- and ion-exchange chromatography under dissociation conditions (buffer containing urea, SDS or guanidine) for the separation of domains proved inefficient due both to insignificant difference in molecular masses and strong aggregation. A selective covalent but reversible immobilization of one of the domains might have proved an efficient approach to their separation. We have chosen for the purpose covalent chromatography of thiol-disulphide exchange commonly used for isolation of proteins containing a free SH-group, as well as for the isolation of SH-containing peptides after the exhaustive hydrolysis of the protein tested by proteolytic enzymes [16-18]. Full account has been taken of the following data: (i) the results of carboxymethylation with iodo[14C]acetic acid showing that domain I contains one cysteine residue whereas domain II contains three residues [1]; (ii) the analytic SDS-electrophoresis in polyacrylamide gel without 2-mercaptoethanol, revealing the absence of interdomain disulphide bond; and (iii) that only cysteine residue may be associated with the haem iron (cysteine being postulated as an axial ligand for different types of cytochromes P-450 by a number of workers [19]), since domain I contains the haem group. Hence, selective immobilization of domain II on the activated thiol-Sepharose, appeared to be the most feasible (see Materials and Methods). In Fig. 2 a scheme for the separation of domains I and II is shown, and in Fig. 3 results from SDS-gel electrophoresis of individual

fractions as well as of the native P-450~ and P-450(I + II) are presented. It can be seen that chromatography on thiopropyl-Sepharose separates domains I and II (Fig. 3, electropherograms C and D, respectively); domain I is eluted by the buffer containing 7 M guanidine that causes the dissociation of the domain complex, and domain II is eluted after the addition of 2-mercaptoethanol. The yields of domains were 80-90% as calculated for P-450(I + II) employed. Since the trypsinolysis of P-450~ afforded 70-80% of P-450(I + II), upon its cleavage, 0.72-0.56 /~mol of individual fragments could be obtained from 1 /~mol of native P-450s~.

Identification in domain H of peptides containing the surface cysteine residues The spectrophotometric analysis showed that in the process of immobilization of both native P450scc and its trypsin-modified form on the activated thiopropyl-Sepharose, 2.0-2.2 ~tmol of

-P-450$c c

~ o

I

I-

u I

B

I--rr~ I

I

I

I

D

+

N~

~ v / P-450(1. IT)

H --

I

0 Domain T. ~--G-S-(~

2-Mercapt°ethan°l = ~ - - S H + ( ~ S N Domain "IT

Fig. 2. Scheme for separation of domains 1 and II of P-450~ using covalent chromatography on activated thiopropyI-Sepharose.

I

3.5

I I I 7 0 3.5 Distance from origin (cm)

4I 7

Fig. 3. Densitometertracings of SDS-polyacrylamidegels after electrophoresis of P-450~ (A), P-450(I+ II) (B) and of individual domains I (C), and II (D) isolated by covalent chromatography on activated thiopropyl-Sepharose.Approx. 20 pg protein were loaded on each gel containing 10% acrylamide.Gels were stained with Coomassiebrilliant blue G-250 and scanned at 550 nm with a Spccord UV-VISspcctrophotometer,equipped with a gel-scanningdevice.

380

2-thiopyridone per 1 #mol protein is released. Hence, the covalent immobilization is achieved at the expense of two exposed SH groups. To simplify the experiments on isolation of cysteine-containing peptides localized in domain II, P-450~c was immobilized, since the interactions of the native molecule and P-450(I + II) with activated thiopropyl-Sepharose have the same characteristics. Analytical variants of hydrolysis of P-450~ immobilized on thiol-Sepharose with trypsin, pepsin and chymotrypsin showed that for the preparation of cysteine-containing peptides, chymotrypsin is the most suitable enzyme. The separation of cysteine-containing peptides was performed on the Servachrom Si 100 C8 phase (Fig. 4). Two homogeneous peptides were obtained from the separation, both having N-terminal amino acid residues of carboxymethylcysteine. The amino acid sequences of peptides containing the surface cysteine residues were the following: CMCys-LeuLeu and CMCys-Val-Gly-Arg-Arg-Ile-Ala-GlnLeu.

70 0.6

///

T i _o

35 o

<

6o

~o

~o Elution volume (ml)

Fig. 4. Purification of surface cysteine-containing peptides of P-450s,~ on a column (0.8 x 25 cm) with Servachrom Si phase 100 C8 (10/~m). Elution was carried out by a linear gradient of acetonitrile from 0 to 70% (v/v) in 0.1% aqueous trifluoroacetic acid. Flow rate, 40 ml/h. Fractions (A,B) shown by solid bars were pooled and subjected to amino acid sequence analysis. Peak A was identified as peptide CMCys-Leu-Leu and peak B as peptide CMCys-Val-Gly-Arg-Arg-lle-Ala-Gln-Leu.

Localization of domains I and H in the polypeptide chain of P-450sc c

According to Table II, the amino acid composition of P-450,~ and the sum of amino acid com-

TABLE II AMINO ACID COMPOSITION OF NATIVE P-450scc AND ITS INDIVIDUAL DOMAINS I AND II Values are residues per molecule Amino acid Asx Thr Ser Glx Pro Gly Ala 1/2Cys Met Ile Leu Tyr Phe Lys His Trp Arg Val Total m o u n t of r~idues Mr

Domain I 20.18 (20) 10.02 (10) 12.11 (12) 27.70 (28) 17.12 (17) 12.67 (13) 10.30 (10) 1.11 (1) 3.53 (4) 12.25 (12) 21.16 (21) 11.55 (12) 14.62 (15) 14.50 (15) 6.82 (7) 5.62 (6) 13.92 (14) 12.53 (13)

Domain II 18.02 (18) 9.94 (10) 9.72 (10) 20.64 (21) 9.94 (10) 9.19 (9) 10.05 (10) 3.49 (3) 8.85 (9) 11.03 (11) 22.50 (23) 5.24 (5) 9.17 (9) 10.59 (11) 4.37 (4) 2.40 (2) 10.92 (11) 12.01 (12)

Sum I + II 38 20 22 49 27 22 20 4 13 23 44 17 24 26 11 8 25 25

P-450scc 36.60 (37) 22.01 (22) 23.83 (24) 48.71 (49) 24.95 (25) 21.62 (22) 20.43 (20) 3.66 (4) 8.79 (9) 25.42 (25) 42.06 (42) 14.97 (15) 25.42 (25) 26.61 (27) 11.17(11) 8.08 (8) 23.76 (24) 26.14 (26)

230

188

418

415

27 000

22 000

-

49 000

381 Domain I (Mr, 2 7 0 0 0 )

/

A

\

NH2- I l e - S e r - T h r - L y s - T h r - P r o - A r g -Pro-

- I l e - P h e - T y r - Gin -Asp- Leu -A rg-

t

Domain 1T ( M r , 22 0 0 0 )

/

A

\

- Arg- Lys-Thr- Glu- Phe-Arg - A s n - T y r - P r o - Gly-

- P r o - Pr~-GIn-Ala-COOH

Fig. 5. Scheme of limited uNpsinolysisof native P-450scc to domains I and II; their localiTatjon in the polypeptide chain of haemoprotein molecules.Thick arrows showthe most trypsin-sensitivepeptide bonds.

positions of domains I and II differs insignificantly, which confirms our assumption of a short trypsin-sensitive polypeptide chain connecting the fragments [1]. When studying the primary structure of P-450s¢¢ we have established the N-terminal amino acid sequence of protein [20] (Fig. 5). The study of individual fragment I showed that its N-terminal sequence (Table III) is identical with the analogous characteristics of the native P-450~c. Consequently, fragment I containing the haem group is the N-terminal moiety of the protein polypeptide chain. Fragment II is the C-terminal moiety of the P-450~ molecule, since the results obtained from the determination of the C-terminal amino acid sequence of P-450~.¢ and those for domain II proved identical (Table III). The analysis of the amino acid sequence of individual fragments I and II (data not presented), in particular, the determination of the C- and

N-terminal amino acid sequences of fragments 1 and II, respectively, established that the site of the amino acid sequence Gln-Asp-Leu-Arg-Arg-LysThr-Glu-Phe (peptide C-XXVIII from chymotryptic hydrolyzate of carboxymethylated P-450~ [21]) is a trypsin-sensitive loop, connecting domains I and II. Discussion In the early works on isolation of the haem-containing peptides from various types of cytochrorues P-450, protein digestion with BrCN was used with subsequent chromatographic purification in formic acid [22,23]. Further attempts to find these peptides in the polypeptide chain of P-450~ were not a success, due probably to their heterogeneity. Recently, the photoaffinity labeling procedure of different types of cytochromes P-450 has been realized to locate the haem-containing peptides

TABLE III AMINO ACID SEQUENCE OF N- AND C-TERMINALSITES OF P-450~ AND ITS INDMDUAL DOMAINS Sample e-450~

N-terminal sequence NH 2-Ile-Ser-Thr-Lys-Thr-Pro-Arg-Pro-

C-terminalsequence -Pro-Pro-Gln-Ala-COOH

Domain I

NH 2-Ile-Ser-Thr-Lys-Thr-

-Ile-Phe-Tyr-Gln-Asp-Leu-Arg-COOH -Ile-Phe-Tyr-Gln-Asp-Len-Arg-Arg-COOH

Domain II

NH 2-Arg-Lys-Thr-Glu-Phe-Arg-Asn-Tyr-Pro-GlyNH 2-Lys-Thr-Glu-Phe-Arg-Asn-Tyr-Pro-GlyNH 2-Thr-Glu-Phe-Arg-Asn-Tyr-Pro-GlyNH 2-Asn-Tyr-Pro-Gly-

-Pro-Pro-Gln-Ala-COOH

382

[24]. However, these were not found in the polypeptide chain of haemoproteins. More recent theoretical analyses of the known primary structure of cytochromes P-450c~m and P-450pB allow differentiation of two homologous sequences apparently responsible for the formation of a haem-binding site [2]. One of these sequences is localized in the N-terminal moiety of cytochromes, and the other in the C-terminal one. Each sequence contains one cysteine residue. A higher degree of homology observed for the Cterminal sequence led the authors to propose that this zone is responsible for binding of the haem group. In the present investigation, as different from literature evidence, it has been shown that the haem group is associated with domain I, the Nterminal moiety of the polypeptide chain (230 amino acids) of P-450s~. The localization of the haem group within this polypeptide fragment I will be the subject of further investigations. The method developed here for the separation of domains I and II on thiopropyl-Sepharose is characterized by two peculiar features. (i) The preparations of P-450scc to be used for the trypsinolysis and subsequent separation should not be subjected to a lengthy storage before use. During storage of the samples of P-450s~ the covalently bound aggregate forms are produced, as is the case with the formation of P-450cam dimer [25]. (ii) Immobilization of P-450(I + II) should be carried out in buffer B, i.e., under conditions of disaggregation of P-450~¢¢ to protometers [20]. The fulfilment of these conditions provided the reproducibility and high yields of the domains. The application of thiopropyl-Sepharose proved unambiguously the absence of a covalent [S-S] bond between the domains, i.e., the rigid interactions of domains are provided by the noncovalent interactions [1]. According to the data from the amino acid analysis, P-450~ and its individual domains I and II contain four, one, and three cysteine residues, respectively (Table II). The isolation of the cysteine-containing peptides of P-450~ using the activated thiopropyl-Sepharose showed that of four possible cysteine-containing peptides, the two SH-peptides identified by us are the surface ones and are localized in domain II. The data on the amino acid sequence of a

number of peptides isolated from enzymatic hydrolyzates of individual domains, and characterizing the C-terminal moiety of fragment I and the N-terminal moiety of fragment II (Table III), enable us to describe the pathway of the limited trypsinolysis of the native P-450s~ (Fig. 5). Initially, there proceeds the cleavage of one peptide chain Arg-Arg (path I) or Arg-Lys (path II). If the first pathway of limited trypsinolysis is realized, the fragment II formed undergoes further hydrolysis with cleavage of the N-terminal dipeptide Arg-Lys, and to a lesser extent the cleavage of the Arg-Asn peptide bond occurs. In the case of second pathway of fragmentation, the domain II formed undergoes further insignificant hydrolysis with removal of the N-terminal pentapeptide. Thus, fragment I, resulting from the incubation of the native P-450scc with trypsin (25°C, 30 min) [1], is a mixture of two polypeptide chains, one of them containing an additional arginine residue with the C-terminal moiety; fragment II is in fact a mixture of four polypeptide chains, whose formation may be schematically presented as gradual cleavage of the N-terminal moiety of the authentic molecule of amino acid fragment, either dipeptide or hexapeptide.

Acknowledgement The help of Mrs. N.L. Petrova in the preparation of the manuscript is gratefully acknowledged.

References 1 Chashchin, V.L.~ Vasilevsky, V.I., Shkumatov, V.M. and Akhrem, A.A. (1984) Biochim. Biophys. Acta 787, 27-38 2 Goton, O., Tagashira, Y., Iizuka, T. and Fujii-Kurijama, Y. (1983) J. Biochem. 93, 807-813 3 Dus, K., Carey, D., Goewert, R. and Swanson, R.A. (1977) in Microsomes, Drug Oxidations (Ullrich, V., ed.), pp. 95-102, Pergamon Press, Oxford 4 Dus, K. (1976) in Enzymes of Biological Membranes (Martonosi, A., ed.), Vol. 4, Part A, Ch. 7, pp. 199-238, Plenum, New York 5 Grassetti, D.R. and Murray, J.F. (1967) Arch. Biochem. Biophys. 119, 41-49 6 Crestfield, A.M., Moore, S. and Stein, W.H. (1963) J. Biol. Chem. 238, 622-627 7 Weber, K. and Osborn, N. (1969) J. Biol. Chem. 244, 4406-4412 8 Thomas, P.E., Ryan, D. and Lewin, W. (1976) Anal. Biochem. 75, 168-176

383 9 Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1205 10 Moore, S. (1963) J. Biol. Chem. 238, 235-237 11 Penke, B., Ferenczi, R. and Kovacs, K. (1974) Anal. Biochem. 60, 45-50 12 Gray, W.R. (1967) Methods Enzymol. 11, 139-151 13 Hayashi, R. (1977) Methods Enzymol. 47, 84-93 14 Gray, W.R. (1967) Methods Enzymol. 11,469-475 15 Yu, C.A. and Gunsalus, I.C. (1974) J. Biol. Chem. 249, 107-110 16 Brooklehurst, K., Carlsson, J., Kierstan, M.P.J. and Crook, E.M. (1973) Biochem. J. 133, 573-584 17 Carlsson, J. and Svenson, A. (1974) FEBS Lett. 42, 183-186 18 Egorov, T.A., Svenson, A., Ryden, L. and Carlsson, J. (1975) Proc. Natl. Acad. Sci. USA 72, 3029-3033

19 Watanabe, T. and Horie, S. (1976) J. Biochem. 79, 829-840 20 Akhrem, A.A., Lapko, V.N., Lapko, A.G., Shkumatov, V.M. and Chashchin, V.L. (1979) Acta Biol. Med. Ger. 38, 257-273 21 Chashehin, V.L., Lapko, V.N., Adamovich, T.B., Lapko, A.G., Kuprina, N.S. and Akhrem, A.A. (1982) Bioorgan. Khim. (Bioorg. Chem. U.S.S.R.) 8, 1307-1320 22 Dus, K., Litchfield, W.J., Mignel, A.G., Hoeven, T.A., Hangen, D.A., Dean, W.L. and Coon, M.J. (1974) Biochem. Biophys. Res. Commun. 60, 15-21 23 Dus, K. (1975) Adv. Exp. Med. Bi01. 58, 287-309 24 Swanson, R.A. and Dus, K.M. (1979) J. Biol. Chem. 254, 7238-7246 25 Limpscomb, J.D., Harrison, J.E, Dus, K.MI and Gtmsalus, I.C. (1978) Bioehem. Biophys. Res. Commun. 83, 771-778