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L A Novikova

Publications and source records attributed to L A Novikova.

At least 19 recordsLinked to original sources
PubMed2006

Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells.

Using the pTrc99A/P450scc vector, a plasmid was constructed in which cDNAs for cytochrome P450scc, adrenodoxin reductase, and adrenodoxin are situated in a single expression cassette. This plasmid was shown to direct the synthesis of all the above proteins in Escherichia coli. Their localization in the E. coli cells and stoichiometry were determined. Cell homogenates exhibited cholesterol hydroxylase/lyase activity, due to catalytically active forms of all three proteins. Thus, the full set of constituents of the mammalian cholesterol hydroxylase/lyase system was shown to be synthesized in bacterial cells for the first time.

Adrenodoxin↗
PubMed2006

Expression of cytochrome P450scc in Escherichia coli cells: intracellular location and interaction with bacterial redox proteins.

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.

Adrenal Cortex↗
PubMed2000

Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Adrenodoxin↗
PubMed1999

Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins.

ATP-Dependent Proteases↗
PubMed1998

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system.

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

ATP-Dependent Proteases↗
PubMed1998

Research of new AgX crystal forms by transmission electron microscopy.

The research of AgX (X=Cl, Br) crystals grown from ammonia solution as a function of X- supersaturation, gelatin concentration, temperature, and use of growth modificators, has been enhanced by data from transmission electron microscopy. The study resulted in preparing AgBr thin films with the most developed ¿100¿ side of rombododecahedron. The size of the crystals was 5 x 15 mm. The thickness was several microm. The new forms of AgBr and AgCl microcrystals were obtained from a high concentrated solution of the corresponding salts in NH4OH. These microcrystals were named "X," 'Y' crystals [AgBr] and stereostructures [AgCl] deriving from their external shapes. As a result of the variation of crystallization parameters, six various morphological types of stereostructures, distinguishable by the size of a central element in relation to external elements and a length of a binding element, were seen. Experiments on photolysis of the stereostructures at the substrate temperature 25-200 degrees C were carried out in vacuum. Exposure to mercury lamp light resulted in characteristic location of large particles of photolytic silver on the stereostructure surface, depending on substrate temperature.

Ammonia↗
PubMed1997

Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast.

A plasmid for effective expression of recombinant DNA encoding a hybrid protein composed of the N-terminal targeting presequence of subunit IV of yeast cytochrome c oxidase preceding the mature polypeptide chain of bovine cytochrome P450scc (pCoxIV-CYP11A1) in yeast has been constructed. It has been shown that this protein, when synthesized in yeast cells, in imported into mitochondria and undergoes proteolytic processing, thus yielding a product of molecular mass corresponding to that of mature cytochrome P450scc. However, only insignificant portion of the imported protein proves to be inserted into the inner membrane of heterologous mitochondria. The membrane-bound cytochrome P450scc exhibits cholesterol hydroxylase activity towards 22R-hydroxycholesterol in the presence of exogenous adrenodoxin and adrenodoxin reductase. This fact indicates that the foreign protein is correctly folded and oriented in the membrane. Thus, insertion into the inner membrane is a limiting step of the pCoxIV-CYP11A1 topogenesis in yeast cells, whereas its import into mitochondria and proteolytic processing proceed without significant impediments.

Animals↗
PubMed1996

Modification of the targeting presequence of the bovine cytochrome P-450scc precursor lifting tissue-specific restrictions on its mitochondrial import.

It has been found that a recombinant cytochrome P-450scc precursor supplemented with an extra MRGSH6GIR sequence at the NH2-terminus (6His-pP450scc) is imported into isolated rat liver and heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing of the precursor resulting in the mature size form of cytochrome P-450scc. Modification of the targeting presequence responsible for its increased positive charge is supposed to lift the previously suggested tissue-specific restrictions on the pP450scc import into mitochondria.

Adrenal Cortex↗
PubMed1995

[Import of a modified form of a cytochrome P450scc precursor into mitochondria from various sources].

An Escherichia coli strain providing hypersynthesis of a recombinant cytochrome P450scc precursor supplemented with the extra MetArgGlySerHis6GlyIleArg sequence at the NH2-terminus (6His-pP450scc) has been constructed. A procedure for isolation and purification of 6His-pP450scc from the cell homogenate has been elaborated. It has been found that the recombinant precursor is imported into isolated rat liver and rat heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing resulting in the mature size form of cytochrome P450scc. Modification of the targeting P450scc presequence resulting in its increased positive charge is supposed to relieve tissue-specific restrictions on the P450scc import into mitochondria.

Amino Acid Sequence↗
PubMed1994

A modified form of the cytochrome P450scc precursor: a new approach in studies on protein import into mitochondria.

Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells. A large-scale procedure for isolation and purification of this protein was elaborated. 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form. As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria.

Amino Acid Sequence↗
PubMed1994

Import of the mammalian cytochrome P450 (scc) precursor into plant mitochondria.

According to previous reports (Matocha M. F. and Waterman M. R. (1984) J. Biol. Chem. 259, 8672-8678 and (1986) Arch. Biochem. Biophys. 250, 456-460) import of the cytochrome P450 (scc) precursor into mitochondria is tissue-specific. The present paper shows that in vitro synthesized bovine cytochrome P450 (scc) precursor can be imported into isolated soybean cotyledon mitochondria and processed therein to the mature size product. This shows that heterologous import of the cytochrome P450 (scc) precursor is possible and that import into mitochondria of this precursor is not restricted to steroidogenic tissues.

Animals↗
PubMed1994

[Design of heterologous mitochondria: import of cattle cytochrome P-450SCC precursors in plant mitochondria].

It has been shown that pre-P-450scc of bovine adrenal cortex mitochondria synthesized in a rabbit reticulocyte lysate cell-free system, is translocated into isolated soybean cotyledon mitochondria, thereby taking the mature form size. This finding is suggestive of the occurrence of a specific receptor and maturase for pre-P-450scc in plant mitochondria. Thus, plant mitochondria can be used as recipients for the mammalian cholesterol hydroxylase system in an attempt to study the mechanism of its formation and preservation.

Adrenal Cortex↗
PubMed1989

Synthesis and secretion of bacterial alpha-amylase by the yeast Saccharomyces cerevisiae.

Alpha-amylase from Bacillus amyloliquefaciens, synthesized in yeast Saccharomyces cerevisiae without substitution of the signal sequence, is efficiently secreted from yeast cells: 60-70% of the overall amount of the enzyme is found in the culture fluid. In contrast to many yeast secretory proteins, which accumulate in the periplasmic space and in the cell wall, intracellular alpha-amylase is localized mainly in the cytoplasm. Obviously, transfer across the cell wall is not a rate-limiting step in alpha-amylase export from the cell. The glycosylated forms of proteins are predominantly found both inside the cell and in the culture medium.

Bacillus↗
PubMed1989

[The prognosis of the visual results of treating children with congenital cataracts based on visual evoked potentials].

Examination of visual induced potentials on reversible chess-pattern in 46 children with bilateral congenital zonular cataracts before and after surgical intervention has revealed correlation between these potentials and degree of lens opacification. After cataract extraction, positive dynamics of the potentials was observed in case of slightly opacified lenses and was absent in intensive lens opacification. The authors consider it reasonable to use visual induced potentials for prognosticating visual results after surgical treatment of bilateral congenital zonular cataracts, this being of particular importance for early surgical interventions.

Cataract↗
PubMed1984

Physical state of the mitochondrial inner membrane as a factor controlling the proteolysis of mitochondrial translation products in yeast.

The degradation of mitochondrial translation products has been studied in Saccharomyces cerevisiae yeast. A high rate of degradation is observed in the early exponential phase of aerobic growth. Maturation of the yeast and glucose repression suppress the degradation. Anaerobic growth is also marked by a low breakdown rate of mitochondrial translation products. These variations did not correlate with the cytochrome c hydrolase activity of sonic submitochondrial particles (this activity was shown to reflect the general state of the proteolytic system of the inner mitochondrial membrane that is responsible for the breakdown of mitochondrially made polypeptides; see Novikova, L.A., et al. (1981) FEBS Lett. 135, 245-248). Experiments with lipid-soluble paramagnetic probes revealed significant variations in the physical state of the mitochondrial inner membrane, as judged from the comparison of the temperature-dependence plots of structural parameters obtained from the EPR spectra of the probes. The breakdown of mitochondrial translation products was, in general, the more rapid the lower was the temperature of the structural transition in the mitochondrial inner membrane and the higher was the relative content of unsaturated fatty acyl chains in the membrane phospholipids.

Electron Spin Resonance Spectroscopy↗
PubMed1983

Correlation between the rate of proteolysis of mitochondrial translation products and fluidity of the mitochondrial inner membrane in Saccharomyces cerevisiae yeast. Alteration of the rate of proteolysis under glucose repression.

Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids.

Cycloheximide↗