Abstract
A procedure for the purification of NADP-linked glyceraldehyde-3-phosphate dehydrogenase from autotrophically grown Euglena gracilis is described. The NADP activity has been purified 103-fold over crude extracts with a 30-fold purification of the NAD activity. A second NAD-dependent activity is eliminated by salting out. The specific activity observed in a typical preparation was (in μmoles hr-1 mg protein-1): 3280 (NADPH) or 510 (NADH). Kinetics were linear and the apparent Km values of the substrates under specified conditions were (μM): NADP, 2.6; NAD, 41.6; NADPH, 18.5; NADH, 7.9; G-3P (NADP) 1000; G-3P (NAD), 120. The ratio VNADP/VNAD of the «oxidative« reaction was 0.7 and the corresponding VNADPH/VNADH ratio of the «reductive« reaction was 5.5. Glyceraldehyde-3-phosphate dehydrogenase enzymes of Euglena either NADP- or NAD-linked, have a molecular weight approximately 150,000 independent of the presence or absence of pyridine nucleotides. Under some conditions the NADP-dependent activity of the purified preparation can be completely lost with little or no decrease of NAD-dependent activity. There is, however, some evidence of kinetic interactions between the two pyridine nucleotide-linked activities. The possibility is discussed that a single protein may catalyze the minor NAD-linked activity of the purified enzyme, as well as the NADP-linked one.
Published Version
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