Biomedical subjects

S Usanov

Publications and source records attributed to S Usanov.

5 recordsLinked to original sources

PubMed2003

A pathway for the metabolism of vitamin D3: unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1).

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.

Adrenodoxin↗

PubMed2001

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae.

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.

Bacterial Proteins↗

PubMed1996

Cytochrome P450scc spin state transitions in the thin solid films.

Langmuir-Blodgett films of cytochrome P450scc were prepared on the solid supports and their spectral properties were investigated. Being immobilized, hemoprotein changes its spin state from initially high to low spin. This transition is reversible since after the solubilization of hemoprotein, the spin state equilibrium is shifted towards high-spin state. Anaerobic reduction of film incorporated cytochrome P450scc by electron transfer chain (NADPH-->adrenodoxin reductase-->adrenodoxin) revealed the low rate of the reaction that coincides well with the content of the hemoprotein low-spin form. We suggest that particularly regular orientation of solid cytochrome P450scc are of crucial importance for this phenomenon.

Cholesterol↗

PubMed1994

Selective chemical modification of Cys264 with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1).

Cys264 of cytochrome P450scc (CYP11A1) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. The labelling affected the interaction of P450scc with adrenodoxin and significantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteoliposomes both the labelled and unlabelled hemeproteins were susceptible to trypsin and split into F1 and F2, two fragments corresponding to the two main domains of P450scc. These results suggest that the hinge connecting the two domains in the region Arg250-Asn257 is exposed to the surface of the membrane and involved in the interaction of P450scc with adrenodoxin.

Adrenodoxin↗

PubMedDate not supplied

Direct visualization of a cardiolipin-dependent cytochrome P450scc-induced vesicle aggregation.

Cytochrome P450scc can be reconstituted successfully into large unilamellar phospholipid vesicles by a combined octylglucoside dialysis/adsorption method. Freeze-fracture electron microscopy was used to analyze the morphology, distribution, and protein topology of the cytochrome P450scc vesicles in dependence on lipid composition. Particles were observed only in close contact to the vesicle surface, probably representing tightly associated cytochrome P450scc at the outer vesicle surface. In cytochrome P450scc vesicles similar in lipid composition to the inner membrane of bovine mitochondria direct evidence by freeze-fracturing was found for a specific cytochrome P450scc-induced aggregation of the vesicles. The vesicle aggregation critically depends on the content of the specific mitochondrial membrane constituent cardiolipin. The aggregation and thus the intervesicular contacts were observed to be inhibited by both addition of anti-cytochrome P450scc IgG and adrenodoxin. Enzymatic reduction of cytochrome P450scc in the liposomal membrane by its electron transfer partners completely indicates an asymmetrical localization in/at the outer side of the bilayer membrane. It is suggested that vesiculation of the inner mitochondrial membrane may be a consequence of the characteristic cardiolipin-dependent cytochrome P450scc membrane topology: the cardiolipin binding, peripheral, non-bilayer-spanning integration as an oligomer in the outer leaflet of the membrane may play a role in the dynamics of formation and dissociation of intramitochondrial vesicles with a functional importance for steroidogenesis.

Adrenodoxin↗