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A number of different dehydrogenases have been shown to bind to Sepharose-bound N(6)-(6-aminohexyl)-AMP. These dehydrogenases can be specifically eluted by binary adducts of NAD(+) or with cofactor gradients. In such manner pure enzymes can be obtained from crude extracts, as demonstrated in the purification on a preparative scale of lactate dehydrogenase from dogfish muscle. The data presented indicate the usefulness of general ligands as affinity agents. The techniques are particularly adaptable for the isolation of human mutant enzymes in blood or in the purification and concentration of enzymes present at low levels in fluids or tissues, as shown in the extensive purification of serum lactate dehydrogenase and glucose 6-phosphate dehydrogenase from hemolysate. lsoenzymes with different affinities for co-enzymes can be separated by affinity techniques. Application of affinity techniques may lead to the separation of isoenzymes or mutant enzymes that are not separable by electrophoretic methods.

Catecholamines bound covalently to glass beads have been found to have biological activity in several systems. Experimental evidence has been found that immobilized epinephrine and isoproterenol accelerate the heart rate in dogs, chick embryo, and chick heart cells grown in culture, whereas immobilized propranolol results in a decrease in heart rate. Isoproterenol bound to glass beads has also been shown to markedly increase the level of adenosine 3':5'-cyclic monophosphoric acid in glial cells. The effects of the immobilized catecholamines are of longer duration than when the compounds are administered in solution. The present data indicate that the compounds are exerting their action when bound to the beads.

Isoproterenol, norepinephrine, and epinephrine covalently bound to glass beads exert a positive inotropic effect on isometrically contracting papillary muscles from cats. Immobilized isoproterenol maintains increases in force and velocity of contraction for more than 5 hr. 1 muM Cocaine potentiates the action of immobilized norepinephrine, isoproterenol, and epinephrine, but not of isoproterenol in solution. The data presented indicate that the effects of immobilized catecholamines are not due to their coming off the glass. The effects observed with cocaine and immobilized catecholamines are not altered by prior treatment of the muscle with reserpine. These results suggest that the major site of catecholamine action is on receptors located on the extended surface of myocardial cells and a post-junctional site for cocaine potentiation.