Abstract
An NAD(+)-specific glutamate dehydrogenase (NAD-GDH) that is inducible by L-glutamine was isolated from Achlya klebsiana and purified to electrophoretic homogeneity. The enzyme is only partially active in vitro unless NADP+ (an activator) is present in both its oxidative deamination and reductive amination reactions. This type of enzyme was reported (LéJohn, H.B. (1971) Nature 231, 164-168) to be widespread among the amorphous group of algae-related fungi classified as Oomycota. The enzyme retained its dependence on NADP+ at all stages of its purification. NADP+ decreased the Km of substrates 3-fold and increased the Vmax 4-fold. M(r) of the undernatured enzyme was 480,000, and, denatured, only a single subunit of M(r) 120,000 was seen. A polyclonal antibody raised in rabbit against purified enzyme subunit excised from SDS-polyacrylamide gel electrophoresis gels immunoprecipitated the M(r) 120,000 polypeptide, the undenatured enzyme, and a physically distinct polypeptide of M(r) 74,000. The antibody, purified against the M(r) 120,000 enzyme subunit as anchored antigen on Sepharose, still immunoprecipitated the M(r) 74,000 polypeptide. The M(r) 74,000 polypeptide was found to be a subunit of a M(r) 220,000 native protein.
Highlights
PURIFICATION AND IMMUNOLOGICAL ANALYSIS*The enzymeis only partially activein vitro un- weight ligands such as purine nucleotides (ADP and AMP), less NADP' (an activatori)s present in botihts oxidative Ca2+,and Mn2+.By and large, it is the animal and fungal GDHs deamination and reductive amination reactions
From the Wanitoba Institute of Cell Biology, Winnipeg, Manitoba R3E OV9,$Department of Microbiology, University of Manitoba Winnipeg, Manitoba R3T 2N2, and Wepartment of Human Genetics, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada
SpectrophotometricNAD-GDH activity was monitored spectrophotometrically by measuring, a t 340 nm, eitherthe rate of reductive amination of a-ketoglutarate with NADH as coenzyme or the rate of oxidative deamination of glutamate with NAD+ as coenzyme as described previously[7].In most instances, the allosteric activator NADP+ was included in thereaction mixture for either the reductive amination oroxidative deamination reaction
Summary
The enzymeis only partially activein vitro un- weight ligands such as purine nucleotides (ADP and AMP), less NADP' (an activatori)s present in botihts oxidative Ca2+,and Mn2+.By and large, it is the animal and fungal GDHs deamination and reductive amination reactions. This that have shown this type of allosteric control [3]. SpectrophotometricNAD-GDH activity was monitored spectrophotometrically by measuring, a t 340 nm, eitherthe rate of reductive amination of a-ketoglutarate with NADH as coenzyme or the rate of oxidative deamination of glutamate with NAD+ as coenzyme as described previously[7].In most instances, the allosteric activator NADP+ was included in thereaction mixture for either the reductive amination oroxidative deamination reaction. The reaction was terminated by exhaustively rinsing the gel with distilled water
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