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Biomedical subjects

M A Eisenberg

Publications and source records attributed to M A Eisenberg.

At least 19 recordsLinked to original sources
PubMed1982

Purification and properties of the biotin repressor. A bifunctional protein.

Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities. The repressor protein can activate biotin in the presence of ATP to form biotinyl-5'-adenylate, the co-repressor which remains tightly bound to the repressor protein. This complex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-CoA carboxylase. The two activities were coincident throughout a purification procedure which resulted in a 3500-fold increase in activity. Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent Mr = 34,000. On gel exclusion chromatography, the activity was also associated with a protein of Mr varying fro 37,000-44,000, indicating the protein is monomeric. The occasional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms. On chromatofocusing, the repressor activity and the holoenzyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point. Only a single protein band with Mr = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.

Acetyl-CoA Carboxylase↗
PubMed1982

Mode of action of the biotin antimetabolites actithiazic acid and alpha-methyldethiobiotin.

Actithiazic acid and alpha-methyldethiobiotin inhibited the conversion of dethiobiotin to biotin resting-cell suspensions of Escherichia coli. The concentrations which effected 50% inhibition were 0.45 and 1.1 microM for actithiazic acid and alpha-methyldethiobiotin, respectively. Cells grown in low concentrations of the two biotin antimetabolites showed derepression of the biotin A operon, as evidenced by the enhanced levels of the enzymes 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase. Derepression was not due to any direct regulatory effect of the antibiotics but was the consequence of the inhibition of the biotin synthetase enzyme; this inhibition prevented the intracellular concentration of biotin from reaching the levels required for normal regulation of the biotin A operon.

Anti-Bacterial Agents↗
PubMed1979

Biotinyl 5'-adenylate: corepressor role in the regulation of the biotin genes of Escherichia coli K-12.

A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for half-maximal binding (K0.5) was calculated to be 1 microM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (K0.5, 10 nM) and much more effective than biotin in the DNA-binding assay (K0.5 1.1 nM versus 1 microM). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.

Adenosine Monophosphate↗
PubMed1978

In vitro synthesis and and regulation of the biotin enzymes of Escherichia coli K-12.

The synthesis and regulation of two of the enzymes of the biotin operon of Escherichia coli, 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, were studied in vitro in a coupled transcription-translation system. These enzymes are encoded by genes located on opposite strands of the divergently transcribed operon (A. Guha, Y. Saturen, and W. Szybalski, J. Mol. Biol. 56:53-62, 1971). The kinetics of synthesis of both the enzymes were determined and the efficiency of the system was 0.3 to 0.4% that of the in vivo rate of synthesis in derepressed cells. Guanosine 3'-diphosphate 5'-diphosphate at 0.2 mM concentration stimulated the synthesis of 7,8-diaminopelargonic acid aminotransferase two- to threefold but had no effect on dethiobiotin synthetase synthesis. Biotin, which was most effective as the corepressor in vivo, also functioned in vitro at physiological concentrations in conjunction with a crude repressor protein isolated from a lysogen carrying the bioR gene. However, the two strands showed differential repression. At a repressor concentration where 7,8-diaminopelargonic acid aminotransferase synthesis was completely repressed, the repression of dethiobiotin synthetase was only 20% and did not exceed 50% with increasing repressor concentrations. Although the exact reason for the partial repression remains to be resolved, our data clearly suggest that the biotin operon is regulated from two separate operators.

Biotin↗
PubMed1975

Purification and properties of 7, 8-diaminopelargonic acid aminotransferase.

The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homogeneous by the scanning of of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values. The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incubation with the amino donor, S-adenosyl-L-methionine. A molecular weight estimation of 94,000 plus or minus 10, 000 has been obtained by gel filtration and sucrose gradient sedimentation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47, 000 plus or minus 3, 000 indicating a dimeric enzyme. A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equivalent to the 7, 8-diaminopelargonic acid produced in the reaction mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5'-methylthioadenosine and the neutral compound, 2-oxo-3-butenoic acid.

Amino Acids, Diamino↗
PubMed1975

Biosynthesis of 7, 8-diaminopelargonic acid from 7-keto-8-aminopelargonic acid and S-adenosyl-L-methionine. The kinetics of the reaction.

The transamination of 7-keto-8-aminopelargonic acid by 7, 8-diaminopelargonic acid aminotransferase of Escherichia coli requires S-adenosyl-L-methionine as the amino donor. Initial velocity studies of this reaction revealed a parallel pattern of reciprocal plots characteristic of a ping-pong mechanism. m-Keto-8-aminopelargonic acid showed strong substrate inhibition which was competitive with S-adenosyl-L-methionine. The Michaelis constants determined for S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid were 0.20 mM and 1.2 muM, respectively. The Vmax of 0.16 mumol/mg/min corresponds to a turnover number for the enzyme of only 17 molecules/molecule enzyme/min. The Km values for the interaction of pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate with the apoenzyme were determined to be 32 muM and 21 muM, respectively. Two classes of inhibitors were observed: (a) those which showed competitive inhibition with respect to S-adenosynd (b) those which showed noncompetitive inhibition with respect to both substrates. In the former group were S-adenosyl-L-(2-hydroxy-4-methylthio)butyric acid and adenosine. In the latter were S-adenosyl-L-ethionine, adenine, and 8-keto-7-aminopelargonic acid. L-Methionine, S-methyl-L-methionine, inosine, and hypoxanthine were not significantly inhibitory. Certain conformations of the substrates in the active site of the enzyme have been proposed which explain: (a) the requirement for the sulfonium ion of S-adenosyl-L-methionine for activity but not for binding to the enzyme, and (b) the ability of 7-keto-8-aminopelargonic acid to bind to the pyridoxal form of the enzyme as a potent substrate inhibitor.

Amino Acids, Diamino↗
PubMed1975

Mode of action of alpha-dehydrobiotin, a biotin analogue.

Alpha-Dehydrobiotin, like biotin, represses coordinately the 7,8-diaminopelargonic acid aminotransferase and the dethiobiotin synthetase enzymes that are encoded on the l and r strands, respectively, of the bioA operon. The rate of synthesis for both enzymes is inhibited about 80% in the presence of alpha-dehydrobiotin. Homobiotin and alpha-methylbiotin are less effective than alpha-dehydrobiotin in repressing the synthesis of the two enzymes. The selective repression of transcription from l and by alpha-dehydrobiotin and homobiotin, previously reported in hybridization experiments, is not observed at the enzyme level. A combination of equal concentrations of biotin and alpha-dehydrobiotin which was reported to enhance selectively the level of messenger ribonucleic acid transcribed from the l strand does not increase the rate of synthesis of the aminotransferase enzyme. Instead, the enzymes encoded on both strands are essentially completely inhibited as with biotin alone. Strain differences have been ruled out to account for the different results obtained by the two methodologies. Our evidence would suggest that alpha-dehydrobiotin acts like biotin, presumably as the co-repressor, in the repression of the bioA operon. The low rates of enzyme synthesis observed in the presence of the biotin analogue is the result of incomplete repression due to a lower affinity of either the analogue for the repressor or of the co-repressor/repressor complex for the operator. While our evidence would support the concept of a two promoter/operator complex, both would have to respond equally to biotin and its analogues. The evidence, however, does not rule out other possible alternative models for the regulation of the biotin operon.

Amino Acids↗
PubMed1974

Active transport of biotin in Escherichia coli K-12.

The transport of [(14)C]biotin into cells of a biotin prototroph, Escherichia coli K-12 strain Y10-1, was investigated. The vitamin taken up by the cells in this strain existed primarily in the free form. Addition of glucose enhanced the rate of uptake six- to eightfold and the steady level was reached in 2 to 3 min resulting in accumulation of biotin against a concentration gradient. The uptake showed marked dependence on temperature (Q(10), 2.3; optimum, 37 C) and pH (optimum 6.6) and was inhibited by iodoacetate. Energy of activation for glucose-dependent uptake was calculated to be 16,200 cal per mol. The rate of biotin uptake with increasing biotin concentrations showed saturation kinetics with an apparent K(m) and V(max) values of 1.4 x 10(-7) M and 6.6 pmol per mg of dry cells per min respectively. The cells also accumulated biotin against a concentration gradient in the absence of added glucose, although at a much lower rate. This accumulation was much more susceptible to inhibition by azide and uncouplers of oxidative phosphorylation suggesting that the energy source was supplied through the electron-transport chain. Inhibition studies with a number of biotin analogues indicated the requirement for an intact ureido ring. The biotin uptake was inhibited in cells grown in biotin-containing medium and was shown to be the result of repression of the transport system, suggesting the control of the biotin transport.

Antimetabolites↗
PubMed1971

Biosynthesis of 7,8-diaminopelargonic acid, a biotin intermediate, from 7-keto-8-aminopelargonic acid and S-adenosyl-L-methionine.

Resting cells of Escherichia coli strain D302(bioD302) can synthesize 7,8-diaminopelargonic acid from 7-keto-8-aminopelargonic acid. The product of this aminotransferase reaction has been identified by paper chromatography and electrophoresis. Glucose enhances the vitamer yield twofold. Of the 19 amino acids tested as amino donors, only methionine proved to be significantly stimulatory. In cell-free extracts, however, methionine was completely inactive unless both adenosine triphosphate (ATP) and Mg(2+) were present. S-Adenosyl-l-methionine (SAM) was about 10 times more effective than methionine, ATP, and Mg(2+). The optimal conditions for the reaction were determined, and substrate inhibition was found for 7-keto-8-aminopelargonic acid. It has been possible to eliminate certain impurities as amino donors in the commercial preparation of SAM and those that may arise in enzymatic reactions in which SAM is a substrate. The direct participation of SAM in the aminotransferase reaction seems a likely possibility.

Adenosine↗