[Use of immobilization for investigation of glyceraldehyde 3-phosphate dehydrogenase. Immobilized tetramers]

Abstract

Glyceraldehyde-3-phosphate dehydrogenase from yeast and rat skeletal muscle was covalently linked to CNBr-activated Sepharose 4B. When the activation was as high as 4-5 mg of CNBr per ml of Sepharose, the enzymes had their maximal activity and were linked to the carrier only by one of the four subunits. The specific activity of immobilized dehydrogenases makes up to 50-60% of that of soluble preparations, since the rate of the substrate diffusion into Sepharose granules is too low. The Km values for NAD and substrate and the pH dependence of the immobilized enzymes were determined. It was found that the enzymes used as adsorbents for isolation of specific antibodies reveal their maximal activity when CNBr concentration reaches 150-200 mg per ml of gel. No inhibition of activity of the immobilized dehydrogenase from yeast or stabilizing effect of antibodies on the enzyme structure were observed.