Some aspects of formation and functioning of the cholesterol hydroxylase system were studied. A hybrid protein was synthesized in E. coli composed of the modified form of the (NADPH)adrenodoxin reductase precursor (N-terminal domain) and the shortened adrenodoxin precursor (C-terminal domain). The modified reductase precursor contained 12 extra amino acid residues at the N-terminus and the N-terminally shortened adrenodoxin precursor had 17 C-terminal amino acids of its targeting presequence. The hybrid reduced cytochrome P450scc in a reconstituted system. Thus, neither the extra 44 amino acids at the N-terminus of the reductase nor the 17 amino acid linker affected the interaction of the active sites in the hybrid protein. These modifications do not interfere with the binding of prosthetic groups and formation of the active sites of two enzymes in the E. coli cells. Modified N-terminal sequence of the hybrid does not affect its import into heterologous mitochondria.
Diethylpyrocarbonate inactivated rat skeletal muscle AMP deaminase in 10 mM phosphate buffer, pH 6.5, at 23 degrees with the second order rate constant of 580 M-1.min-1. Absorbtion at 240 nm was concomitantly increase. Enzyme activity can be restored by hydroxylamine. The pH-dependence of inactivation indicates the involvement of a group with pKa 6.9. The data suggest that modification of one histidyl residue per subunit inactivates the activity of tetrameric AMP deaminase.
The effect of micromolar concentration of rhodamine 123 (methylrhodamine) and ethyl and amyl esters of unsubstituted rhodamine on oxygen consumption by rat liver mitochondria was studied under irradiation by an argon laser (488 and 514 nm). Irradiation of mitochondria in the presence of rhodamine stimulates their respiration. Light-induced stimulation of respiration is not inhibited by free radical scavenger ionol and by inhibitor of the permeability transition pore cyclosporine A. Stimulation of respiration by moderate doses of radiation is reversed in the dark. Increase in radiation dose resulted in only partial reversal of stimulated respiration in the dark. Rhodamine efficacy in stimulation of mitochondrial respiration depends on its structure (amyl > ethyl > methylrhodamine).
Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.
Pepsin secretion was studied in vivo in the primary culture of isolated secretory gastric mucosa cells of the frog (Rana ridibunda). The injection of animals with carbacholin (200 micrograms/100 g weight) or histamine (100 micrograms/100 g weight) or addition of the hormones (100 microM) into the incubation medium of isolated gastric cells significantly increased pepsin activity. In vitro, preliminary addition of the specific antagonists of these hormones to the incubation medium (1 microM atropine or 100 microM cimetidine) completely suppressed the effects of hormones. The effect of histamine is season-dependent. Combined injection or simultaneous addition to incubation medium of isolated cells of carbacholin and histamine caused mutual potentiation of their effects. In this case, the quantitative response of the cells exceeded the theoretical sum of the stimulatory effects of each of these two hormones alone. According to electrophoresis, the hormones cause quantitative changes in isozyme spectrum of proteolytic enzymes but do not significantly affect their qualitative characteristics.
A site-specific endonuclease which recognizes the sequence 5'-CCTNAGG-3' was purified to homogeneity from the thermophilic strain Bacillus sp. R7. The endonuclease (BspR7I) is monomeric protein with an apparent molecular weight of 37 kD. The enzyme is active over a wide range of NaCl concentrations, pH, and temperatures. BspR7I cleaves DNA substrates according to the scheme: 5'-CC decreases TNAGG-3' 3'-GGANT increases CC-5', hence the endonuclease represents an isoschizomer of Bsu361.
Activities of basic carboxypeptidases (CP) in rat brain regions and peripheral tissues were determined on postnatal days 0-90. The highest phenylmethylsulfonyl fluoride-inhibited activity was detected immediately after birth; activity decreases on day 10 with subsequent increase on day 20 and decrease on day 90. The CPE activity was the lowest immediately after birth, increased on day 30, and decreased on day 90. The lysosomal CPB activity was highest at birth and on day 90 and was the lowest on day 20. The activity of CPN slowly increases till day 20 and then it decreases. The relationship between dynamics of basic CP activity in ontogenesis and functional role of these enzymes is discussed.
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The effects of the Rhodiolae rosea extract on contractility of rat hearts isolated by the Langendorff's technique, creatine phosphokinase activity in the perfusate, and induction of myocardial stress protein synthesis (hsp-70) were studied after total ischemia and reperfusion. The adaptogen produces cardioprotector effects and prevents ischemia- and reperfusion-induced contractility aberrations. The possible role of inducible stress proteins (hsp-70) in these effects is discussed.
The series of nitronylnitroxyl radicals (NNR) were studied as paramagnetic scavengers of nitric oxide. These radicals react with NO with rate constants of (0.6-1.1).10(4) M-1.sec-1 forming stable iminonitroxyl radicals. They can be used to assay nitric oxide in solutions by EPR spectroscopy; the sensitivity of the method is 1 microM for the detection of NO concentration and 0.3 nM/sec for the measurements of the rates of NO generation for 1 h in 0.2 ml sample. To overcome fast reduction of the radicals in biological samples, charged NNR was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes thus decreasing the rates of NNR reduction about 1000-fold. The method was used for the NO synthase activity assay in rat cerebellum cytosol. NNR was used to study the kinetics of the decomposition of 3,4-dihydro-1,2-diazete 1,2-dioxides (DD). Several DD derivatives at 5-80 microM concentrations are very effective vasodilators in perfused rat tail artery. Intraperitoneal injection of several DD (40-200 micrograms/kg weight). in hereditary hypertensive rats (ISIAH-strain) significantly (by 30%) decreased systolic arterial blood pressure whereas similar effect of trinitroglycerin was detected at significantly higher dose (900 micrograms/kg weight).
The major cytoplasmic mRNP protein of somatic cells, p50, is the member of the Y-box-binding transcription factor family and can control gene expression at two levels including mRNA transcription and translation. It has been demonstrated that p50 is responsible for the inactive state of mRNA within free mRNPs. In this work, we show that the Y-box-containing DNA (Y-DNA) predominantly binds to p50 in rabbit reticulocyte lysates and causes translation inhibition of the endogenous and exogenous globin mRNA and prokaryotic beta-galactosidase mRNA. Preincubation of Y-DNA with purified p50 prevents the inhibition. Inhibition of protein biosynthesis by the Y-DNA is not due to the degradation or functional inactivation of mRNA. The inhibition is associated with the decay of polyribosomes and dissociation of a newly synthesized protein from the ribosomes. The data indicate that Y-DNA inhibits protein biosynthesis predominantly at the initiation stage and that p50 is an essential component of the translation initiation apparatus.
The kinetics of hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine catalyzed by hemin and hemin-bovine serum albumin (BSA) complex (methemalbumin) was studied in buffered physiological solution containing dimethylformamide (40%) and dimethyl sulfoxide (2%), respectively. The formation of hemin-BSA complex enhanced hemin catalytic activity in oxidation of both amines. The reaction follows first-order kinetics in biocatalyst concentrations, H2O2, and H+ ions from pH 6.5 to 9.0. The catalytic constants, Michaelis constants, and kcat/K(m) ratios for both substrates in hemin- and methemalbumin-catalyzed reactions were calculated using double reciprocal plots of the effect of the initial TMB and PDA concentrations on the initial reaction rates. Mechanism of radical oxidation of amines in hemin-H2O2 and hemin-BSA-H2O2 systems is discussed. Both systems can effectively initiate radicals free radicals formation; their activity is similar to the previously studied ferritin-H2O2 system.
Activities of RNA polymerases from wild type B/r strain of E. coli and its pleiotropic rpoB403 rif-r mutant were studied using five various DNA matrices and various groups of early region promotors of T4 and T7 phage DNA. The mutation differentially affects RNA polymerase-catalyzed RNA synthesis depending on the promotor used. Relative efficiency of A3 promotor of T7 phage DNA is enhanced in case of the mutant enzyme. rpoB403 mutation can be used for the classification analysis of promotors differing in their interaction with the enzyme.
Antiaggregatory properties of guanidine thiol derivatives and their effect on human platelet guanylate cyclase activity were studied. The molecules of guanidine thiols contain guanidine and thiol groups which are the donor and acceptor of nitric oxide (NO), respectively. Three synthetic guanidine thiol derivatives were studied including mercaptoethylguanidine (MEG), mercaptoethylguanidine disulfide (MEG-disulfide), and mercaptoethylguanidine methylated at SH-group (S-methylmercaptoethylguanidine (S-methyl MEG)). All compounds are the substrates of NO-synthase and activators of human platelet guanylate cyclase. The stimulatory effects of MEG and MEG-disulfide on guanylate cyclase activity were 2- and 4-fold higher, respectively, than the effect of L-arginine. Stimulation of the enzyme by S-methyl MEG is similar to L-arginine. Antiaggregatory properties of these compounds correspond to the extent of guanylate cyclase activation. Extent of guanylate cyclase activation (S-methyl MEG < MEG < MEG-disulfide) significantly correlates with inhibition of ADP-induced platelet aggregation and with acceleration of spontaneous disaggregation of platelets. The mechanism of directed enhancement of antiaggregatory properties of the compounds can depend on their chemical structure and extent of guanylate cyclase activation.
The review summarizes data on structure, allosteric regulation, and denaturation of muscle glycogen phosphorylase b. Specific attention is paid to correlations between the structure and function of phosphorylase b and molecular mechanism of its allosteric regulation. Chemical and thermal denaturation of phosphorylase b is reviewed.
The polyphosphatase with specific activity of 283 units/mg was purified 3450-fold to homogeneity with 3.8% yield from cytosol of Saccharomyces cerevisiae yeast. Polyphosphatase is monomeric 40 kD protein. The enzyme hydrolyzes polyphosphates of various chain length including tripolyphosphate but ATP, pyrophosphate, and p-nitrophenyl phosphate are not the substrates. Enzyme activity is maximal at 50 degrees C and pH 6.5-8.5. Several cations of bivalent metals stimulated the enzyme activity 8-66-fold (Co2+ > Mn2+ > Mg2+ > Zn2+ > Fe2+). The enzyme is inactive in the presence of Ca2+ or Cu2+. Heparin, antibodies against cell-envelope polyphosphatase, and Cu2+ or Zn2+ in the presence of Mg2+ are potent inhibitors of cytosolic polyphosphatase. Cytosolic polyphosphatase is similar to purified cell-envelope polyphosphatase but differs in some properties from nuclear, vacuolar, and mitochondrial polyphosphatase of the very same yeast.
Level of malonic dialdehyde, lipid peroxidation, and activities of main antioxidant enzymes were studied in respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae yeast. The data confirm the hypothesis of V.P. Skulachev that the respiratory system is directly involved in the antioxidant defense of the cells.
A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.
Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.