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Biomedical subjects

A T Brown

Publications and source records attributed to A T Brown.

8 recordsLinked to original sources
PubMed1979

Regulation of lactose catabolism in Streptococcus mutans: purification and regulatory properties of phospho-beta-galactosidase.

Phospho-beta-galactosidase (P-beta-gal), the enzyme which catalyzes the first step in the metabolism of intracellular lactose phosphate, occurred at high specific activity in the cytoplasm in 12 of 13 strains of streptococcus mutans grown on lactose but not other carbon sources. The P-beta-gal from S. mutans SL1 was purified 13-fold using diethylaminoethyl-cellulose ion exchange and agarose A--0.5 M molecular exclusion column chromatography. The molecualr weight of the enzyme was estimated to be 40,000, and its pH optimum was 6.5 in three different buffer systems. P-beta-gal activity was inhibited by Co2+, Zn2+, and Cu2+, but other cations, ethylenediaminetetraacetic acid, orthophosphate, and fluoride had no effect upon enzyme activity. The kinetic response of P-beta-gal to a model substrate, o-nitrophenyl-beta-D-galactopyranoside-6-phosphate, obeyed Michaelis-Menten kinetics, and the Km for this substrate was 0.19 mM. In addition to being under genetic control, P-beta-gal activity was regulated by a number of biologically active metabolites. Enzyme activity was inhibited in a sigmoidal fashion by phosphoenolpyruvate. The M 0.5 V value for phosphoenolpyruvate was 2.8 mM, and the Hill coefficient (n) was 3. In addition, P-beta-gal exhibited strong inhibition by ATP, galactose-6-phosphate, and glucose-6-phosphate. In contrast to inhibition of P-beta-gal activity by phosphoenolpyruvate, the inhibition exerted by ATP, galactose-6-phosphate, and glucose-6-phosphate obeyed classical Michaelis-Menten kinetics; the Ki values for these inhibitors were 0.55, 1.6, and 4.0 mM, respectively.

Adenosine Triphosphate↗
PubMed1977

Polyol metabolism by a caries-conducive Streptococcus: purification and properties of a nicotinamide adenine dinucleotide-dependent mannitol-1-phosphate dehydrogenase.

The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii) diethyl-aminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at -80 degrees C in the presence of beta-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenedia-minetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NAD-dependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)-dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate and NAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K(m) values for mannitol-1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K(m) values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine 5'-triphosphate and fructose-6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.

Alcohol Oxidoreductases↗
PubMed1977

Comparative study of invertases of Streptococcus mutans.

Sucrase activity was studied in 13 strains of Streptococcus mutans representing the five Bratthall serotypes. Sucrose-adapted cells have sucrase activity in the 37,000 x g-soluble fraction of all strains. The enzyme was identified as invertase (beta-d-fructofuranoside fructohydrolase; EC 3.2.1.26) because it hydrolyzed the beta-fructofuranoside trisaccharide raffinose, giving fructose and melibiose as its products, and because it hydrolyzed the beta-fructofuranoside dissacharide sucrose, giving equimolar glucose and fructose as its products. Invertases of c and e strains exhibit two activity peaks by molecular exclusion chromatography with molecular weights of 45,000 to 50,000 and about 180,000; those of serotypes a, b, and d strains exhibit only a single component of 45,000 to 50,000 molecular weight. The electrophoretic mobility of invertases is different between the serotypes and the same within them. Inorganic orthophosphate (P(i)) has a weak positive effect on the V(max) of invertases of serotypes c and e cells but a strong positive effect on the invertases of serotype b cells; P(i) has a strong positive effect on the apparent K(m) of the invertases of serotype d cells, but has no effect on the V(max); P(i) has a strong positive effect on both the apparent K(m) and V(max) of the invertases of serotype a cells. Thus, the invertases were different between all of the serotypes but similar within the serotypes. These findings support the taxonomic schemes of Coykendall and of Bratthall. It was additionally noted that 37,000 x g-soluble fractions of only serotypes b and c but not serotypes a, d, and e cells have melibiase activity, and it could be deduced that serotype d cells lack an intact raffinose permease system.

Cell-Free System↗
PubMed1975

Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase from Actinomyces viscosus.

A nicotinamide adenine dinucleotide-specific L-(+)-lactate dehydrogenase (LDH) (EC 1.11.27) from Actinomyces viscosus T-6-1600 was purified approximately 110-fold by a combination of diethylaminoethyl-cellulose and 0.5 M Agarose A column chromatography. The ldh was stable at 26 C, but was quite labile at temperatures below 5 C. The enzyme had a molecular weight of 100,000 +/- 10,000 as determined by 0.5 M Agarose molecular exclusion chromatography and showed optimum activity between pH 5.5 and 6.2. The A. viscosus LDH exhibited homotropic interactions with its substrate, pyruvate, and its coenzyme, reduced nicotinamide adenine dinucleotide, indicating multiple binding sites on the enzyme for these ligands with some degree of cooperative interaction between them. The enzyme was under negative control by adenosine 5'-triphosphate, and its kinetic response to the negative effector was sigmoidal in nature. Inorganic phosphate reversed the inhibition exerted on the A. viscosus LDH by adenosine. The 5'-triphosphate thermal stability at 65 C of the LDH from A. viscosus was increased in the presence of its negative effector, adenosine 5'-triphosphate, but was markedly decreased in the presence of its coenzyme, reduced nicotinamide adenine dinucleotide. The glycolytic intermediate, fructose-1,6-diphosphate, had no effect on the catalytic activity of the A. viscosus LDH at saturating pyruvate concentrations. However, fructose-1,6-diphosphate was a potent positive effector at low substrate concentrations. Thus the A. viscosus LDH is under positive control by fructose-1,6-diphosphate and inorganic phosphate, but under negative control by adenosine 5'-triphosphate.

Actinomyces↗