Glutamate Dehydrogenase from Mycoplasma laidlawii

Abstract

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.

Structure of NADP+‐dependent glutamate dehydrogenase from Escherichia coli – reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

“…Despite their overall structural similarities, microbial GDHs have distinct cofactor preferences. The enzymes can be subdivided into three groups, depending on their preference for NAD + , NADP + , or both almost equally [10][11][12]. Analyses of sequences and structures of other nicotinamide cofactor-dependent enzymes have led to various explanations for the molecular basis of cofactor specificity.…”

Glutamate dehydrogenase from Escherichia coli: purification and properties

“…They were stored in 7% acetic acid. Enzymatic activity in the gels was detected by staining as described by Yarrison et al (20).…”

NADP-dependent glutamate dehydrogenase from a facultative methylotroph, Pseudomonas sp. strain AM1

“…Denaturing SDS-electrophoresis revealed a minimum molecular weight of 50,000, indicating that the enzyme is composed of four subunits. GDH enzymes from E. coli (23) and Bacillus megaterium (13) have six subunits, and that from Mycoplasma laidlawii has five (28).…”

“…Coprinus cinereus (1), Hydrogenomas sp. strain H16 (16), Thiobacillus novellus (17), and Pseudomonas aeruginosa (15) possess both NAD-and NADP-dependent enzymes, and Mycoplasma laidlawii has a GDH of dual coenzyme specificity (28…”