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M A Levy

Publications and source records attributed to M A Levy.

70 records · Page 4Linked to original sources
PubMed1989

Interaction between rat prostatic steroid 5 alpha-reductase and 3-carboxy-17 beta-substituted steroids: novel mechanism of enzyme inhibition.

Efforts to identify novel compounds capable of blocking the steroid 5 alpha-reductase (SR) catalyzed conversion of testosterone (T) to 5 alpha-dihydrotestosterone have resulted in the development of 17 beta-substituted-3-androstene-3-carboxylic acids as potent inhibitors of the rat prostatic enzyme. The dead-end inhibition patterns of one of these steroidal acrylates, 17 beta-N-(2-methyl-2-propyl)-carbamoyl-androst-3,5-diene-3-carboxylic acid were best evaluated with a linear uncompetitive kinetic model vs both T (Kii = 11 +/- 1 nM) and NADPH (Kii = 22 +/- 2 nM). To interpret these observations, the kinetic mechanism of the rat prostatic SR was shown to involve the binding of NADPH prior to that of T through a series of dead-end and product inhibition experiments. Within the context of this preferentially ordered kinetic mechanism, it is proposed that the uncompetitive inhibition patterns result from the association of the steroidal acrylate to an enzyme complex containing NADP+ in formation of a dead-end ternary complex of enzyme, NADP+, and inhibitor.

5-alpha Reductase Inhibitors↗
PubMed1989

Inhibitors of steroid 5 alpha-reductase in benign prostatic hyperplasia, male pattern baldness and acne.

Benign prostatic hyperplasia is an androgen-dependent disease which afflicts a large percentage of males over the age of fifty, and is usually treated by surgery. Dihydrotestosterone, a 5 alpha-reduced metabolite of testosterone, has been implicated as a causative factor in the progression of the disease, largely through the clinical study of males who are genetically deficient in the dihydrotestosterone-producing enzyme, steroid 5 alpha-reductase. As a result, inhibition of this enzyme has become a pharmacological strategy for the treatment of benign prostatic hyperplasia as well as other dihydrotestosterone-related disorders such as acne and male pattern baldness. In this review, Brian Metcalf and colleagues focus on the chemical and kinetic mechanisms of steroid 5 alpha-reductase, and known inhibitors of this enzyme, and discuss the rationale behind the design of a mechanistically distinct class of steroid 5 alpha-reductase inhibitors.

5-alpha Reductase Inhibitors↗
PubMed1989

The irritancy and allergenicity of 2-n-octyl-4-isothiazolin-3-one (Skane M-8), with recommendations for patch test concentration.

Preparations of 2-n-octyl-4-isothiazolin-3-one (Skane M-8) at 1000 p.p.m. a.i. and 500 p.p.m. a.i. in pet. induced allergy in a cumulative insult human patch test. Challenge patch testing using 250 p.p.m. a.i. Skane M-8 elicited allergy in 6 out of 6 sensitized individuals. Sensitized individuals did not cross react with Kathon biocide, (Kathon CG), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. All pretrolatum preparations to which Tween 85 was added at 2.5% produced mild irritation in cumulative insult patch tests, but no irritation was observed with Tween 85 at 0.625%. We conclude that Skane M-8, 250 p.p.m. a.i. with Tween 85 0.625% in petrolatum is appropriate for patch testing for Skane M-8 allergy.

Adult↗
PubMed1988

Ultrastructural and biochemical analysis of the stress granule in chicken embryo fibroblasts.

The ultrastructure and biochemical composition of cytoplasmic particles that form in chicken embryo fibroblasts during stress have been analyzed. We showed previously that these particles contained the small stress protein, sp 24, and antibodies specific to sp 24 were used here to identify the stress granule. In thin sections, the stress granule was a densely staining, membraneless, cytoplasmic body and appeared as a highly condensed area of cytoplasm in freeze-fracture preparations. Hypotonic swelling of cells before freeze-fracture analysis revealed a basketlike structure composed of interconnecting protein cables. No other proteins could be cross-linked to sp 24 when stress granules were treated with dithiobis-(succinimidyl propionate). High resolution autoradiographic analysis with [3H]uridine failed to identify any associated RNA synthesized in the period immediately before the stress. Thus the stress granule appears to be composed predominantly of sp 24 aggregates. Sp 24 could be purified to homogeneity from the stress granule by solubilization in 8 M urea and anion exchange chromatography.

Amino Acids↗
PubMed1987

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles.

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindström, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗
PubMed1987

Substrate stereospecificity and active site topography of gamma-aminobutyric acid aminotransferase for beta-aryl-gamma-aminobutyric acid analogues.

The substrate and inhibitory properties of (R)- and (S)-4-amino-3-phenylbutanoic acid, (R)- and (S)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofens), (E)-4-amino-3-phenylbut-2-enoic acid, and (E)-4-amino-3-(4-chlorophenyl)but-2-enoic acid are determined and compared with those of 4-aminobutanoic acid, 4-aminobut-2-enoic acid (4-aminocrotonic acid), and the racemic mixtures of 4-amino-3-arylbutanoic acids. All compounds in both series were found to be substrates, except for the R-isomers, which were identified as competitive inhibitors. These results are compared with known pharmacological data regarding the appropriate isomers.

4-Aminobutyrate Transaminase↗
PubMed1985

Morphological analysis of ligand uptake and processing: the role of multivesicular endosomes and CURL in receptor-ligand processing.

The receptor-mediated endocytosis and intracellular processing of transferrin and mannose receptor ligands were investigated in bone marrow-derived macrophages, fibroblasts and reticulocytes. Mannosylated bovine serum albumin (BSA) conjugated to colloidal gold (Au-man-BSA) or colloidal gold-transferrin (AuTf) were used to trace ligand processing in these cells. These ligands appeared to be processed by mechanisms similar to those observed previously with other mannose receptor and galactose receptor ligand probes. After uptake via coated pits and coated vesicles, Au-man-BSA appeared in small uncoated vesicles and tubular structures and was transferred to large, sometimes multivesicular endosomes (MVEs), which sometimes had arm-like protrusions reminiscent of CURL (compartment of uncoupling of receptor and ligand) [10, 11]. Initially these structures became increasingly multivesicular, but during longer incubations the inclusion vesicles appeared to disintegrate to leave a denser, amorphous lumen. Inclusion vesicle disintegration may result from the introduction of lysosomal enzymes into these structures. These results suggest a model for differential receptor-ligand and ligand-ligand sorting. As suggested [10, 11] membrane constituents may be recycled to the plasma membrane from the arms of CURL. Receptor-bound ligands, such as transferrin, would also recycle. The luminal contents, including dissociated ligands, other soluble proteins and inclusion vesicles (containing some membrane proteins), would target to lysosomes. This would result in the lysosomal degradation of any membrane proteins that were incorporated in the inclusion vesicle membranes.

Animals↗
PubMed1983

Fluorescence decay time measurements of Eu3+-ATP-enzyme complexes. Replacement of the metal hydration water by active site ligands.

Measurements of the fluorescent lifetimes of the rare earth metal Eu3+ in varying mole fractions of H2O/D2O were used to determine the hydration of the metal in the presence of ATP and/or hexokinase or chloroplast reversible ATPase. The number of water molecules coordinated to the metal in Eu3+-ATP was estimated to be 2.6; when this complex is bound to hexokinase, 1 water molecule is displaced. Upon binding to chloroplast reversible ATPase, the metal coordinates 1 water molecule while the Eu3+-ATP complex does not retain any associated solvent. These numbers are in contrast to the 9 solvent molecules coordinated to the naked metal ion. These results are discussed in reference to mechanistic and structural considerations of the two enzymes.

Adenosine Triphosphatases↗
PubMed1983

In vitro and in vivo effects on brain GABA metabolism of (S)-4-amino-5-fluoropentanoic acid, a mechanism-based inactivator of gamma-aminobutyric acid transaminase.

The effects of intraperitoneal administration of (S)-4-amino-5-fluoropentanoic acid, a mechanism-based covalent inactivator of gamma-aminobutyric acid transaminase (GABA-T), on whole brain GABA metabolism in mice were investigated. A dose-dependent and time-dependent irreversible inactivation of GABA-T was observed with a concomitant increase in whole brain GABA levels. The compound exhibited no in vitro nor in vivo time-dependent inhibition of glutamate decarboxylase (GAD), alanine transaminase, or aspartate transaminase (Asp-T). It was, however, a potent competitive reversible inhibitor of GAD and a weak competitive inhibitor of Asp-T. The chloro analogue, (S)-4-amino-5-chloropentanoic acid, was ineffective.

4-Aminobutyrate Transaminase↗
PubMed1983

Metal and sulfur composition of iron-molybdenum cofactor of nitrogenase.

The sulfur content of N-methylformamide solutions of cofactor from Clostridium pasteurianum nitrogenase has been determined to be 11.9 (+/- 0.9) mol per mol of molybdenum. This number was determined radiochemically, using iron-molybdenum cofactor isolated from molybdenum-iron protein from bacteria grown on 35SO4. A total of 3.2 (+/- 0.2) mol of sulfur per mol of molybdenum was found to be present in cysteine and methionine, probably arising from contaminating proteins not intrinsic to the cofactor. Combined with accumulated evidence that is discussed, these results lead to an updated stoichiometry of MoFe6S8 or 9, not MoFe6S4 as previously thought, for this cluster.

Bacterial Proteins↗
PubMed1981

Substituted 4-aminobutanoic acids. Substrates for gamma-aminobutyric acid alpha-ketoglutaric acid aminotransferase.

Substituted 4-aminobutanoic acids were studied as potential irreversible inactivators of purified pig brain gamma-aminobutyric acid aminotransferase, the enzyme responsible for the degradation of the inhibitory neurotransmitter, gamma-aminobutyric acid. It was found that unlike the related 4-amino-5-halopentanoic acids (Silverman, R. B., and Levy, M. A. (1981) Biochemistry 20, 1197), the 4-amino-3-halobutanoic acids were substrates for this enzyme, undergoing exclusive elimination to succinic semialdehyde and producing no inactivation. The hydroxy analogue, however, underwent exclusive transamination and no succinic semialdehyde was detected. These results are discussed in terms of the nature of the substituents, the structure of the active site of gamma-aminobutyric acid aminotransferase, and the design of mechanism-based inactivators.

4-Aminobutyrate Transaminase↗
PubMed1981

Mechanism of inactivation of gamma-aminobutyric acid-alpha-ketoglutaric acid aminotransferase by 4-amino-5-halopentanoic acids.

(S)-4-Amino-5-halopentanoic acids were previously shown to irreversibly inhibit pig brain gamma-aminobutyric acid--alpha-ketoglutaric acid aminotransferase, and a mechanism for the inactivation was proposed (Silverman, R. B., & Levy M. A. (1980) Biochem. Biophys. Res. Commun. 95, 250). Evidence is presented to show that these compounds are mechanism-based inactivators, and experiments are described to elucidate their mechanism of action. The enzyme must be in the pyridoxal phosphate form for inactivation to occur, the gamma proton of the inactivators is removed in a rate-determining step, and one fluoride ion is released from 4-amino-5-fluoropentanoic acid per active site labeled. The change in the optical spectrum of the enzyme during inactivation suggests that the coenzyme is converted into the pyridoxamine phosphate form. Every turnover of inactivator leads to an inactivation event, i.e., the partition ratio is zero.

4-Aminobutyrate Transaminase↗
PubMedDate not supplied

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of a novel A-ring pyrazole steroid.

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17 beta-ol (3), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)beta-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a Ki of 90 +/- 20 nM. The activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5 alpha-reductase (from rat prostate), and 3 alpha-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3. Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.

3-Hydroxysteroid Dehydrogenases↗