Biomaterial Database

Comparative structural and immunochemical characterization of recombinant and natural cytochrome p450scc (CYPXIAI). 1998

G I Lepesheva, and S A Usanov

Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Zhodinskaya 5/2, Minsk, 220141 Belarus. usanov@ns.iboch.ac.by

Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc. The quantitative values of the studied parameters are practically identical in natural and substrate-bound recombinant cytochrome P450scc, while there were great differences between substrate-bound and substrate-free forms of recombinant cytochrome P450scc both of functional (decrease of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in the reconstituted system, and affinity to adrenodoxin for substrate-free cytochrome P450scc) as well as structural (increase in accessibility to exogenous and endogenous proteolysis) character. The identity of the folding process for recombinant and natural proteins as well as the nature of a stabilizing and activating effect of cholesterol on cytochrome P450scc is discussed.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008928	Mitochondria	Semiautonomous, self-reproducing organelles that occur in the cytoplasm of all cells of most, but not all, eukaryotes. Each mitochondrion is surrounded by a double limiting membrane. The inner membrane is highly invaginated, and its projections are called cristae. Mitochondria are the sites of the reactions of oxidative phosphorylation, which result in the formation of ATP. They contain distinctive RIBOSOMES, transfer RNAs (RNA, TRANSFER); AMINO ACYL T RNA SYNTHETASES; and elongation and termination factors. Mitochondria depend upon genes within the nucleus of the cells in which they reside for many essential messenger RNAs (RNA, MESSENGER). Mitochondria are believed to have arisen from aerobic bacteria that established a symbiotic relationship with primitive protoeukaryotes. (King & Stansfield, A Dictionary of Genetics, 4th ed)	Mitochondrial Contraction,Mitochondrion,Contraction, Mitochondrial,Contractions, Mitochondrial,Mitochondrial Contractions
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002417	Cattle	Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.	Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D002784	Cholesterol	The principal sterol of all higher animals, distributed in body tissues, especially the brain and spinal cord, and in animal fats and oils.	Epicholesterol
D002786	Cholesterol Side-Chain Cleavage Enzyme	A mitochondrial cytochrome P450 enzyme that catalyzes the side-chain cleavage of C27 cholesterol to C21 pregnenolone in the presence of molecular oxygen and NADPH-FERRIHEMOPROTEIN REDUCTASE. This enzyme, encoded by CYP11A1 gene, catalyzes the breakage between C20 and C22 which is the initial and rate-limiting step in the biosynthesis of various gonadal and adrenal steroid hormones.	CYP11A1,Cholesterol Desmolase,Cholesterol Monooxygenase (Side-Chain-Cleaving),Cytochrome P-450 CYP11A1,Cytochrome P-450(scc),20,22-Desmolase,CYP 11A1,Cytochrome P450 11A1,Cytochrome P450scc,20,22 Desmolase,Cholesterol Side Chain Cleavage Enzyme,Cytochrome P 450 CYP11A1
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005287	Ferredoxin-NADP Reductase	An enzyme that catalyzes the oxidation and reduction of FERREDOXIN or ADRENODOXIN in the presence of NADP. EC 1.18.1.2 was formerly listed as EC 1.6.7.1 and EC 1.6.99.4.	Adrenodoxin Reductase,Iron-Sulfur Protein Reductase,NADPH-Ferredoxin Reductase,Ferredoxin NADP Reductase,Iron Sulfur Protein Reductase,NADPH Ferredoxin Reductase,Protein Reductase, Iron-Sulfur,Reductase, Adrenodoxin,Reductase, Ferredoxin-NADP,Reductase, Iron-Sulfur Protein,Reductase, NADPH-Ferredoxin