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

Abstract

MATERIALS AND METHODS Chemicals. Sodium pyruvate, reduced nicotinamide adenine dinucleotide (NADH), FDP, and ATP were purchased from the Sigma Chemical Co., St. Louis, Mo., as were all other components of the LDH assay system. Diethylaminoethyl (DEAE)-cellulose (DE 52) was purchased from Reeve Angel, Clifton, N. J., and 0.5 M Agarose A was obtained from BioRad Laboratories, Richmond, Calif. All components of the A. viscosus growth medium were obtained from Difco Laboratories, Detroit, Mich. Growth of organisms. A. viscosus strain T-6-1600 was grown anaerobically at 37 C in a medium composed of tryptone (0.5% wt/vol), yeast extract (0.5% wt/vol), dibasic potassium phosphate (0.5% wt/vol), and Tween-80 (0.05% wt/vol). The medium was supplemented with sucrose (0.5% wt/vol) as the primary energy source. The organism was routinely grown in 6-liter batches of the complex medium described above, and cells were harvested after an incubation period of 16 h at 40 C. All cells were washed once with 0.01 M potassium phosphate buffer, pH 7.0, after harvesting them by centrifugation, and the cell pellets were frozen at-20 C until used. Laboratory cultures of A. viscosus strain T-6-1600 were kept in NIH fluid thioglycolate medium which contained fluid thioglycolate, 2.9% (wt/vol), and beef extract, 1.2% (wt/vol), supplemented with calcium carbonate, 0.2% (wt/vol). Culture transfers were made

Bacterial lactate dehydrogenases.

“…The other type of bacterial L(+)-nLDH, that activated by FDP, is found in most streptococcal species, in a few lactobacilli (notably L. casei), and in a few other bacteria (Table 1). Many of these nLDH's show an absolute requirement for FDP at physiological pH's (46,106), although that of Actinomyces viscosus only requires FDP at low pyruvate concentrations (5). Butyrivibrio fibrisolvens is reported to have an nLDH activated by FDP (94).…”

“…Some workers have preferred carbohydrates other than glucose. L. oenos was grown on ribose (50), S. cremoris was grown on lactose (52), Streptococcus mutans was grown on mannitol (6), and A. viscosus was grown on sucrose (5). Lactic acid bacteria should not be grown aerated; a closed vessel is satisfactory, but some strains have been grown with reduced oxygen (16,46,48,100).…”

Carbon dioxide metabolism by Actinomyces viscosus: pathways for succinate and aspartate production

“…PEPC activity was assayed by monitoring the incorporation of ['4C]NaHCO3 into nonvolatile, acid-stable products. The incubation mixture contained the following components in a final volume of 1.0 ml: phosphoenolpyruvate (PEP), 5.0 Imol; MnCl2, 1.0 jmol; ['4C]NaHCO3, 5 ,mol (1 ,uCi); reduced nicotinamide adenine dinucleotide (NADH), 4 Itmol; pig heart MDH, 5 U; enzyme, 0.1 ml; Tris-maleate, pH 6.5, 50 jmol; and distilled water to volume. The reaction was initiated with A. viscosus PEPC, and the assay mixture was incubated at 370C for 20 min.…”

“…All cells were washed once with 0.01 M potassium phosphate buffer, pH 7.0, and the cell pellets were frozen at -20'C until used. Laboratory cultures of A. viscosus were stored as previously described (1). Preparation of cell suspensions for intact cell experiments.…”

Regulation of invertase of Actinomyces viscosus

“…There is much evidence implicating Actinomyces viscosus in the etiology of both periodontal disease and certain forms of caries in both humans and rodents (6,8,(10)(11)(12). Although the role of this microorganism has been studied in the etiology of these diseases, far less is known about the metabolism of A. viscosus (3,4,7,9,13). Our laboratory has previously reported the presence, separation, and partial purification of two enzymes involved in the initial dissimilation of dietary disaccharides by this microorganism, invertase and /3-galactosidase (13).…”