The Use of Biochemical Solid‐Phase Techniques in the Study of Alcohol Dehydrogenase

Abstract

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr-activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea-treated Sepharose-bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S-polypeptide chain are active when using 5p-dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.Immobilized enzymes have received widespread attention during the last few years. Among the numerous examples of their application found in the literature are reports illustrating the potential of immobilized enzymes as means for investigating more fundamental biochemical problems [l]. In particular, the immobilization technique has been used, to advantage, in probing the relationship between enzyme quaternary structure and enzyme action. Such studies on matrix-bound subunits have provided valuable information regarding whether the quaternary structure is necessary for enzyme activity and about subunit interactions in oligomeric enzymes [2-61. In these investigations, the immobilized subunit was prepared by first coupling the oligomer in such a manner that preferentially a single subunit would be linked covalently to the matrix and then the immobilized preparation was treated with a protein denaturant, such

In vitro dissociation and reassociation of human alcohol dehydrogenase class I isozymes

“…The success of interspecies hybridization of horse liver ADH with human (3^^isozyme suggested structural homology between the horse and the human enzyme (Schenker et al, 1971;Berger et al, 1974) even before the amino acid sequence of the human enzyme was reported. Moreover, by carrying out the dissociation/ reassociation on enzyme attached to a solid support, it was also demonstrated that the monomer of horse liver ADH is inactive (Andersson & Mosbach, 1979).…”

Immobilization of horse liver alcohol dehydrogenase on copolymers of glycidyl methacrylate and ethylene dimethacrylate

“…Some theoretical problems associated with structure and function of alcohol dehydrogenases also have been solved by studying suitably immobilized enzymes. 8 The present work has been aimed at immobilization of horse liver alcohol dehydrogenase (EC 1.1.1.1) (LADH) to glycidyl methacrylate with ethylene dimethacrylate copolymers, and a choice of the most suitable type of the carrier Biotechnology and Bioengineering, Vol. XXIV, Pp.…”