Primary structure of ferredoxin from bovine kidney mitochondria.

Kidney mitochondrial ferredoxin (renodoxin) is a component of the cytochrome P-450-dependent enzymatic system whose main function is the hydroxylation of vitamin D3 in the 1alpha- and 24-positions. The complete amino acid sequence of renodoxin was determined by protein chemistry and mass spectrometry. The mature renodoxin has 128 amino acid residues. The N- and C-terminal regions of renodoxin are subject to proteolytic modification, this being the origin of heterogeneous molecular mass (from 14,200 to 12,400 kD) of purified protein preparations. The antigenic structure of renodoxin was studied using antibodies to peptide fragments of a homologous protein, adrenodoxin.

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.

Chemical modification of rabbit IgG with N-dansylaziridine. Investigation of the properties of dansylated antibodies.

To elucidate the effect of antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ), which is sensitive to the changes in the microenvironment, was used for modification of rabbit IgG hinge region cysteine residue. DAZ binds to hinge region Cys 226 as evidenced by the structural analysis. Labelling of IgG with DAZ does not alter its conformation, hydrodynamic behavior, nor its antigen binding properties. Upon antigen bindings, the fluorescence intensity of modified IgG increases about 80%. This result indicates that interaction of antibodies with antigen is accompanied by the conformational changes in the IgG hinge region.

[Chemical modification of rabbit immunoglobulin G by dansylaziridine and study of the properties of modified antibodies]. / Khimicheskaia modifikatsiia immunoglobulinov G krolika N-dansilaziridinom i issledovanie svoistv modifitsirovannykh antitel.

To elucidate the effect of the antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ) which is sensitive to microenvironmental changes, was used for modification of the rabbit IgG hinge region cystine residue. DAZ binds to the hinge region Cys 226 as could be evidenced from the structural analysis data. Labelling of IgG with DAZ does not alter either its conformation and hydrodynamic behaviour or its antigen binding properties. Upon antigen binding the fluorescence intensity of modified IgG increases up to about 80%. This finding suggests that the interaction of antibodies with the antigen is accompanied by conformational changes in the IgG hinge region.

[Comparative study of bovine adrenodoxin and renorenodoxin]. / Sravnitel'noe izuchenie adrenodoksina i renoredoksina byka.

The ferredoxin from bovine renal mitochondria (renoredoxin) has been obtained in a highly purified state. The A415/A280 ratio of the purified renoredoxin is 0.84. The absorption spectrum of renoredoxin was shown to be identical to that of bovine adrenodoxin. Two forms of renoredoxin (Mr 14200 and 13300) were detected by using polyacrylamide gel electrophoresis. These forms exhibit a very similar immunologic cross-reactivity with polyclonal antibodies to adrenodoxin. The N-terminal amino acid sequence of renal ferredoxin was shown to be identical to that of adrenodoxin; the C-terminal sequences of both ferredoxins undergo a similar post-translational proteolytic modification. The amino acid composition of ferredoxins are also very close. Renal ferredoxin can be replaced by adrenodoxin in reconstituted systems from bovine adrenal cortex mitochondria which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin.

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria.

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.

[A covalently linked complex of cytochrome P-450 with adrenodoxin. Localization of the adrenodoxin binding site of cytochrome P-450]. / Kovalentno sshityi kompleks tsitokhroma P-450 s adrenodoksinom. Lokalizatsiia adrenodoksinsviayvaiushchego uchastka tsitokhroma P-450.

Cytochrome P-450scc and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of a cross-linked complex and 6% of free cytochrome P-450scc was obtained after purification on cholate-Sepharose. Cytochrome P-450scc in the cross-linked complex is not reduced in the presence of NADPH and adrenodoxin reductase, but completely preserves its high spin form in the presence of Tween-20 or pregnenolone. The use of radioactive labelled adrenodoxin, chemical cleavage of cytochrome P-450scc from the cross-linked complex by o-iodosobenzoic acid and HPLC for separation of peptides demonstrated that the cytochrome P-450scc complex with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450scc, i.e., Leu 88-Trp108 and Leu368-Trp417.

[Primary structure of hepatoredoxin from bovine liver mitochondria]. / Pervichnaia struktura gepatoredoksina iz mitokhondrii pecheni byka.

The primary structure of hepatoredoxin from bovine liver mitochondria was established. It consists of 117 amino acid residues. The identity of the amino acid sequences of bovine hepatoredoxin and adrenodoxin from bovine adrenal cortex mitochondria was shown. It is assumed that the role of a ferredoxin component in mitochondrial steroid-hydroxylating systems from different organs is played by the same [2Fe-2S]-protein.

Primary structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria and some aspects of its functioning on a structural level.

The primary structure of the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) from bovine adrenocortical mitochondria has been determined. At the initial stage an exhaustive chymotryptic digestion of carboxymethylated P-450scc was performed, and the amino acid sequence of 66 peptides was determined. At the second stage an investigation of the amino acid sequence of individual fragments I (Mr 29 800) and II (Mr 26 600) of the limited trypsinolysis of P-450scc was carried out. Fragment I was digested with trypsin, Staphylococcus aureus V8 proteinase and thermolysin; fragment II was cleaved with trypsin and S. aureus V8 proteinase. In addition, the amino acid sequence of some CNBr peptides of P-450scc has been investigated. The primary structure of cytochrome P-450scc determined with protein chemistry methods proved the multistage cholesterol transformation to pregnenolone to be catalyzed by a single species of cytochrome P-450scc which consists of 481 amino acids. The results from protein sequencing of P-450scc are in good agreement with those obtained recently from nucleotide sequencing. The localization of peptide bonds cleaved under limited proteolysis of P-450 with trypsin to fragments I and II, I and III (Mr 16 800) is presented. It is shown that the transformation of P-450scc to P-420 is accompanied by the appearance of an additional trypsin-sensitive peptide bond in the N-terminal part of P-450scc.

[Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence]. / Pervichnaia struktura 20S,22R-kholesteringidroksiliruiushchego tsitokhroma P-450 iz mitokhondrii kory nadpochechnikov byka. IV. Struktura peptidov termoliticheskogo i ogranichennogo tripticheskogo gidrolizov fragmenta F1; peptidy bromtsianovogo rasshchepleniia tsitokhroma P-450. Polnaia aminokislotnaia posledovatel'nost'.

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.

[Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. III. Primary structure of peptides produced by hydrolysis of the fragment F2 with proteinase from Staphylococcus aureus]. / Pervichnaia struktura 20S,22R-kholesteringidroksiliruiushchego tsitokhroma P-450 iz mitokhondrii kory nadpochechnikov byka. III. Issledovanie struktury peptidov, poluchennykh pri gidrolize fragmenta F2 proteinazoi iz Staphylococcus aureus.

Primary structure of F2 fragment resulting from limited trypsinolysis of the native cytochrome P-450 has been investigated. Hydrolysis of F2 fragment with proteinase from Staphylococcus aureus afforded 18 homogeneous peptides covering the whole polypeptide chain of the fragment. Complete amino acid sequences were established for 16 peptides, two peptides being elucidated partially. The above data in combination with structural study of chymotryptic peptides of cytochrome P-450 and tryptic peptides of F2 fragment led to reconstitution of six peptide blocks of F2 fragment comprising 203 amino acid residues.

[Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. Study of the structure of peptides obtained by hydrolysis of fragment F1 with proteinase from Staphylococcus aureus]. / Pervichnaia struktura 20S,22R-kholesteringidroksiliruiushchego tsitokhroma P-450 iz mitokhondrii nadpochechnikov byka. II. Issledovanie struktury peptidov, poluchennykh pri gidrolize fragmenta F1 proteinazoi iz Staphylococcus aureus.

The fragment F1 resulting from the limited tryptic hydrolysis of the native molecule of cytochrome P-450 has been digested with Staphylococcus aureus protease. 24 peptides, covering the whole polypeptide chain of fragment F1, are isolated from the hydrolysate. Analysis of their amino acid sequence in combination with the earlier data on the structure of cytochrome P-450 chymotryptic peptides and fragment F1 tryptic peptides permitted to carry out a reconstruction of large peptide blocks of fragment F1 that comprise 252 amino acid residues.

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain.

Cytochrome P-450scc 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-450scc 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 II 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-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.