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At least 145 records · Page 8Linked to original sources
PubMed2005

Synthesis and biological activity of new iodoacetamide derivatives on mutants of squalene-hopene cyclase.

New iodoacetamide derivatives, containing a dodecyl or a squalenyl moiety, were synthesized. The effect of these new thiol-reacting molecules was studied on two mutants of Alicyclobacillus acidocaldarius squalene-hopene cyclase constructed especially for this purpose. In the quintuple mutant, all five cysteine residues of the enzyme are substituted with serine; in the sextuple mutant, this quintuple substitution is accompanied by the substitution of aspartate D376, located at the enzyme's active site, with a cysteine. N-Dodecyliodoacetamide had little activity toward either mutant, whereas N-squalenyliodoacetamide showed a stronger effect on the sextuple than on the quintuple mutant, as expected.

Bacillus↗
PubMed2005

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae.

Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas' disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Using a homogenate of S. cerevisiae cells expressing T. cruzi OSC, we have tested 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, selected as representative of different classes of substrate analog inhibitors of OSC. The IC50 values of inhibition (the compound concentration at which the enzyme is inhibited by 50%) are compared with the values obtained using OSC of pig liver and S. cerevisiae. Many inhibitors of pig liver and S. cerevisiae OSC show comparable IC50 for T. cruzi OSC, but some phenylthiovinyl derivatives are 10-100 times more effective on the T. cruzi enzyme than on the pig or S. cerevisiae enzymes. The expression of proteins of pathogenic organisms in yeast seems very promising for preliminary screening of compounds that have potential therapeutic activity.

Animals↗
PubMed1999

19-Azasqualene-2,3-epoxide and its N-oxide: metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae.

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7+/-2 and 25+/-5 microM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [14C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 microM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and >100 microM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in "ultrasonically stimulated" cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.

Enzyme Inhibitors↗
PubMed1985

Separation by high-performance liquid chromatography of (3R)- and (3S)-beta-leucine as diastereomeric derivatives.

For studies on the coenzyme B12-dependent enzyme, leucine-2,3-aminomutase, (3R)- and (3S)-beta-leucines were synthesized. The 10-camphorsulfonamide p-nitrobenzyl esters could be resolved by normal-phase HPLC. A much better separation was obtained by reversed-phase HPLC of the diastereomeric derivatives obtained by treatment of -leucine with Marfey's reagent (N2-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide).

Alanine↗
PubMed1988

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase.

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase. However, for unknown reasons, no leucine 2,3-aminomutase activity could be found in rat liver homogenate.

Amino Acid Isomerases↗
PubMed1991

Bulk purification of isochorismic acid by low-pressure octadecyl (C18) reverse-phase liquid chromatography.

Partially purified isochorismate synthase (EC 5.4.99.6) from Klebsiella pneumoniae 62-1 was used to produce bulk quantities (3.4-6.8 mg) of isochorismate from chorismate. A new, preparative, low-pressure liquid chromatographic method for the purification of isochorismate was used; a (1.0 X 13.0 cm) octadecyl (C18) reverse-phase column with a discontinuous, stepped methanol gradient as eluent. The recovery of isochorismate was quantitative and its purity was verified by HPLC using a butyl (C4) reverse-phase column. This chromatographic method is superior to those previously described.

Chorismic Acid↗
PubMed1991

Conditions for the assay of glutamate semialdehyde aminotransferase that overcome the problem of substrate instability.

Investigations into the kinetic properties of glutamate semialdehyde aminotransferase, a key enzyme in the metabolic pathway leading to chlorophyll, are made difficult by the instability of the enzyme's substrate glutamate 1-semialdehyde. The rate of spontaneous disappearance of this compound from solution is shown to vary with the square of its concentration and to be pH-dependent. Thus using conditions appropriate to the assay of the enzyme, half of the substrate is lost from solution in a few minutes. Second-order rate constants for the reaction are determined and conditions are selected whereby the effects of the spontaneous reaction are rendered insignificant. The steady-state kinetic properties of the enzyme determined using these conditions are reported.

Chloroplasts↗
PubMed1990

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli.

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.

DNA Transposable Elements↗
PubMed1991

Separation and partial characterization of enzymes catalyzing delta-aminolevulinic acid formation in Synechocystis sp. PCC 6803.

Formation of the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differentiatial ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 microM, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 microM. GSA aminotransferase was inhibited 50% by 0.5 microM gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 degrees C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.

Aldehyde Oxidoreductases↗
PubMed1992

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae.

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.

Detergents↗
PubMed1981

Squalene epoxidase, oxido-squalene cyclase and cholesterol biosynthesis in normal and tumoral mucosa of the human gastro-intestinal tract. Evidence of post-HHG CoA regulation.

Cholesterol biosynthesis was evaluated in different parts of the human gastro-intestinal tract by incorporation of labelled acetic or mevalonic acid into non-saponifiable metabolites and also by measuring squalene epoxidase activity, oxido=squalene cyclase activity and dihydrolanosterol demethylation rate. With respect to rat liver, whole cholesterol biosynthesis was generally low and we found an anatomic localization of activity towards the end of the gastro-intestinal tract. The results of analysis of mevalonic acid metabolites implied the existence of post-HMG CoA regulation. A comparison with the corresponding dat obtained from homologous cancerous tissues (inthe case of high cholesterogenic activity) did not show a greater activity in the tumor, in spite of higher levels of epoxidase concentration.

Acetates↗
PubMed1988

Inhibition of sterol biosynthesis in Saccharomyces cerevisiae by N,N-diethylazasqualene and derivatives.

The ability of some azasqualene derivatives to inhibit yeast cell growth was compared with their inhibition activity on squalene-2,3-oxide cyclase (EC 5.4.99.7) both in living cells and in microsome preparations. Among the compounds tested, N,N-diethylazasqualene showed the best correlation between the activity on squalene-2,3-oxide cyclase and its inhibition of yeast growth. The N-oxide derivative, N,N-diethylazasqualene N-oxide, which was as active as the amine in microsomes, was much less active in living cells, probably because it could not easily penetrate the cell wall. Kinetic analysis of the inhibitory activity of compounds on squalene-2,3-oxide cyclase revealed a sharp difference between N,N-diethylazasqualene and its N-oxide; the former showed a non-competitive-type inhibition, whereas the latter behaved as a competitive inhibitor.

Aza Compounds↗