Improved activity sedimentation method

Yarrison, Gerald; Choules, G.L.

doi:10.1016/0003-2697(72)90427-7

Cited by 3 publications

(2 citation statements)

References 8 publications

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“…As shown in Fig. 2, values of the relative mobility (Rm) of NADP-and NAD-linked enzyme activities were almost the same (Rm of 0.17) in all extracts, indicating that an identical enzyme catalyzed both NADP-and NAD-linked reactions in Bs fragilis as found in B. thetaiotaomicron (7), Selenomonas ruminantium (14), and Mycoplasma laidlawii (15). In addition, the second activity band which was specific for NAD with Rm of 0.25 was found in the extracts from the 50 mM ammonia cells.…”

Section: Enzyme Activities For Ammonia Assimilationmentioning

confidence: 67%

The pathway of ammonia assimilation in Bacteroides fragilis.

Yamamoto

Abe

Saito

et al. 1984

J. Gen. Appl. Microbiol.

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The enzymes in ammonia assimilation pathways have been examined in a strictly anaerobic bacterium Bacteroides fragilis. The activities of NADPH-and NADH-linked glutamate dehydrogenase and glutamine synthetase were demonstrated in extracts of cells, but very low activity of NADPH-dependent glutamate synthase and no activity of alanine dehydrogenase were demonstrated. Both activities of the glutamate dehydrogenase were not distinguished electrophoretically, indicating the presence of a dual pyridine nucleotide-specific enzyme. At low concentrations of ammonia in batch cultures and at low dilution rates of continuous flow cultures, higher activities of glutamate dehydrogenase were found in the cells. The values of Km of NADPH-linked glutamate dehydrogenase were 0.8 mM, 0.15 mM, and 7 uM for ammonia, 2-oxoglutarate, and NADPH, respectively. Although glutamine synthetase activity was also higher in cultures with limited ammonia and at low dilution rates of continuous cultures, this enzyme may not be important for ammonia incorporation into amino acids, since cell growth was not affected by the addition to the culture of methionine sulfoximine, a glutamine synthetase inhibitor. This evidence suggests that ammonia assimilation is mainly carried out by the glutamate dehydrogenase in B. fragilis.

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Section: Enzyme Activities For Ammonia Assimilationmentioning

confidence: 67%

The pathway of ammonia assimilation in Bacteroides fragilis.

Yamamoto

Abe

Saito

et al. 1984

J. Gen. Appl. Microbiol.

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“…The approximate molecular weight of M. laidlawii glutamate dehydrogenase was estimated by a modification of the activity sedimentation method of Cohen (6). Details of our procedure will be published elsewhere (22). We found that the molecular weight was 250,000 i 10%.…”

Section: Fraction (Ml)mentioning

confidence: 99%

Glutamate Dehydrogenase from Mycoplasma laidlawii

1972

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Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H) ]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 + 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The Km for reduced nicotinamide adenine dinucleotide was 1.8 x 10-4 M. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported. MATERIALS AND METHODS Chemicals. a-Ketoglutaric acid, NAD, NADPH, ribonuclease (all A grade) and deoxyribonuclease (B grade) were obtained from Calbiochem. Phenol reagent (Folin-Ciocalteau) was from Fisher Scientific Co. Chemicals for acrylamide gel electrophoresis were all products of Matheson, Coleman, and Bell. Phenazine methosulfate, MTT tetrazolium [3(4,5 dimethyl thiazolyl-2)-2, 5-diphenyl tetrazolium bromide], tris(hydroxymethyl) aminomethane (Tris), NADP (Sigma grade), and NADH (grade 111) were obtained from Sigma Chemical Co. Sepharose 6B and diethylaminoethyl (DEAE) Sephadex A-50 were obtained from Pharmacia Fine Chemicals, Inc. Ammonium sulfate, A.R. was from Mallinckrodt Chemical Works. DEAE cellulose DE.11 was obtained from Reeve Angel, Inc. Crude extract preparation. M. laidlawii, strain A, culture and osmotic lysis procedure were as described previously (4). The organisms were extracted once with 10-3 M ethylenediaminetetraacetic acid (EDTA), pH 7.5, followed by two more extractions with demineralized water. The extracts were then combined and subjected to the enzyme fractionation procedure described later.

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Sedimentation of generalized systems of interacting particles. II. Active enzyme centrifugation—theory and extensions of its validity range

Cohen

Claverie

1975

Biopolymers

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SynopsisThe first detailed application of a recently published very general approach to chemical equilibria during sedimentation is presented. As a consequence of the very extensive theoretical treatment, made possible by this approach, the active enzyme analytical centrifugation method can now be used under a far wider set of conditions than before, including the study of many interacting active molecule systems. It has also been shown that this method is as precise as the more conventional ones.

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Improved activity sedimentation method

Cited by 3 publications

References 8 publications

The pathway of ammonia assimilation in Bacteroides fragilis.

The pathway of ammonia assimilation in Bacteroides fragilis.

Glutamate Dehydrogenase from Mycoplasma laidlawii

Sedimentation of generalized systems of interacting particles. II. Active enzyme centrifugation—theory and extensions of its validity range

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