Isolation and properties of glyceraldehyde-3-phosphate dehydrogenase from a sturgeon from the Caspian Sea and its interaction with spin-labeled NAD + derivatives

Abstract

Glyceraldehyde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate: NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) was isolated from a sturgeon, Huso huso, from the Caspian Sea. It is closely related to the enzyme from a Pacific sturgeon, Acipenser transmontanus, with respect to amino acid composition, steady-state kinetics and coenzyme binding. The latter, as studied by means of a spin-labeled derivative of NAD +, is negatively cooperative exhibiting a Hill coefficient of 0.84 at 12°C. Two derivatives of NAD + spin-labeled at N 6 or C 8 of the adenine ring were found to be active coenzymes with maximum velocities reaching 35 or 45% of the value for NAD + itself. When more than two equivalents of either spin-labeled NAD + are bound to the enzyme spin-spin interactions are observed in the ESR spectra. Distances between the nitroxide radicals (8–9 Å) calculated from the observed splittings are in excellent agreement with data predicted from the crystal structure of the lobster enzyme when the coenzyme is bound in an anti-conformation of the adenine moiety about the glycosidic bond to all four subunits.