Purification and characteristics of an enzyme with both bilirubin oxidase and laccase activities from mycelium of the basidiomycete Pleurotus ostreatus.

A homogenous enzyme with both bilirubin oxidase and laccase activities was isolated from a submerged culture of the basidiomycete Pleurotus ostreatus mycelium and characterized. The yield of the enzyme was 127 microg/g dry biomass of the mycelium. The specific activity of the enzyme was 21 and 261 U/mg to bilirubin and to a laccase substrate ABTS, respectively. The intracellular phenol oxidase from the P. ostreatus mycelium was identified as bilirubin oxidase with the amino acid sequence highly homologous to that of the pox2 gene-encoded product. The enzyme displayed the maximal laccase activity at 50-55 degrees C to all substrates examined, whereas the pH optimum was substrate-dependent and changed from 3.0 for ABTS to 7.0 for syringaldazine and guaiacol. The enzyme maintained catalytic activity within a broad pH range but was inactivated at pH 4.0. The enzyme was thermostable but very sensitive to metal chelating inhibitors. Trypan Blue (5 mg/liter) was completely decolorizated upon 3 h of incubation with the bilirubin oxidase (20 mU/ml) at room temperature.

Modification of transport function of plasma albumin during atherosclerosis and diabetes mellitus.

Parameters characterizing the processes of association, transport, and dissociation of fatty acid molecules on the corresponding binding sites of plasma albumin in patients with atherosclerosis and diabetes mellitus were studied by electron paramagnetic resonance method. In these patients transport function of albumin differed from normal. It should be emphasized that these differences were specific for atherosclerosis and diabetes mellitus, which is of considerable diagnostic value.

Isoforms of thyroxin-binding globulin as markers of ecologically induced pathologies.

The content of thyroxin-binding globulin isoforms differing by the carbohydrate constituent is significantly increased in the blood of coronary patients and patients with insulin-dependent diabetes mellitus compared to normal and is comparable to that in cancer patients. Enhanced biosynthesis of abnormal proteins due to structural and functional reorganization of plasma membranes is probably responsible for accumulation of thyroxin-binding globulin isoforms.

Discrimination between conformational states of mitochondrial cytochrome P-450scc by selective modification with pyridoxal 5-phosphate.

Electrophoretically homogeneous cytochrome P-450scc preparation isolated by the standard method from adrenal cortex mitochondria comprises two protein forms differing in the accessibility of their amino groups to specific chemical modification with pyridoxal 5-phosphate. The protein form whose lysine amino groups are accessible to the modifier constitutes about 60-70% of the preparation. Being covalently bound to pyridoxal 5-phosphate, this protein form loses enzymatic activity and affinity for adrenodoxin. This protein form can be separated by affinity chromatography on adrenodoxin-Sepharose. The cytochrome P-450scc form whose amino groups are not accessible to the modifier is retained on the affinity matrix, and after elution from adrenodoxin-Sepharose has the absorption spectrum typical of the high-spin protein with a spectral homogeneity index A392/A278 = 1.0. The enzymatic activity of the hemoprotein form whose lysine amino groups are inaccessible to the modification is identical to that of the initial unmodified protein.

[Crystallization of a covalently-linked complex of adrenodoxin and adrenodoxin reductase]. / Kristallizatsiia kovalentno-sshitogo kompleksa adrenodoksina i adrenodoksinreduktazy.

Two forms of mitochondrial adrenodoxin reductase from bovine adrenals and recombinant bovine adrenodoxin and adrenodoxin reductase expressed in Escherichia coli were isolated, purified to homogeneity and biochemically characterized. Recombinant adrenodoxin reductase was expressed as a single polypeptide; its retention time on DEAE-Fractogel coincides with the second form (F2) of the mitochondrial reductase. Two enzyme forms have similar adrenodoxin reductase activities in two types of systems comprising either cytochrome c or cytochrome P-450 (11 beta) as the terminal electron acceptor. Adrenodoxin and each of two reductase forms were cross-linked using 1-ethyl-3-(dimethyl-amino-propyl)carbodiimide. An effective two-step method for the purification of the active heterologous cross-linked complexes is suggested that enables purification of the functional complexes to homogeneity. The cross-linked bimolecular complex of adrenodoxin and adrenodoxin reductase was crystallized for the first time.

Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link.

In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed.

[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]. / Khimicheskaia modifikatsiia steroidgidroksiliruiushchikh monooksigenaz fluorestseinizotiotsianatom.

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

[Localization of tetranitromethane-modified tyrosine residues in the polypeptide chain of cholesterol-hydroxylating cytochrome P-450]. / Lokalizatsiia modifitsirovannykh tetranitrometanom ostatkov tirozina v polipeptidnoi tsepi kholesteringidroksiliruiushchego tsitokhroma P-450.

As a continuation of earlier structure-function relationship studies on cholesterol-hydroxylating cytochrome P-450 from the adrenal cortex mitochondria, the present study deals with the distribution of tetranitromethane-modified tyrosine residues in the hemeprotein polypeptide chain. Amino acid residues Tyr-24, -46, -50, -93, -94, -199, -246 are shown to be modified with tetranitromethane. Tyr-93, -94 are supposedly involved in the active site formation of cytochrome P-450.

[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.

Isolation, structural organization and mechanism of action of mitochondrial steroid hydroxylating systems.

A scheme for the isolation of individual proteins of the 20S,22R-cholesterol hydroxylating system is presented. Physico-chemical and structural parameters of cytochrome P-450 are furnished. Self-reconstitution of indivudal proteins of the system into an enzyme unit is shown. Furthermore a regulation process of electron transport in the 20S,22R-cholesterol system is considered.