George J. Koval

Transamination reactions of rat brain

Abstract The spectrum of transamination reactions of rat brain involving α-ketoglutarate and pyruvate have been quantitatively estimated employing an extremely sensitive, fast and simple technique of assay. Evidence is presented in support of the separate identities of enzymes catalyzing the transaminations from phenylalanine, tyrosine, 3,4-dihydroxyphenylalanine, tryptophan and 5-hydroxytryptophan to α-ketoglutarate.

Thin-layer chromatography for the separation of nucleotides

Abstract A fast and simple thin-layer chromatographic method for separating nucleotides and inorganic phosphate for the subsequent quantitative determination of radioactivity is described. DEAE-cellulose is washed with acetic acid before spreading onto glass plates; formic acid is used as the solvent in a closed vessel.

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.

ANALYSIS OF Na+, K+ AND NUCLEOTIDE INTERACTIONS IN TERMS OF A HETEROTROPIC RELAXATION MODEL FOR (Na+‐K+)‐ATPase

Previous studies of the partial reactions of (Na+-K+)-ATPase have tended to support models of active Na+ transport that invoke cycling of the system between two major conformational states. 1-3 The oscillation is driven by forces generated by a cycle of phosphorylation and hydrolysis of an acceptor group that is integral to the enzyme. Since the phosphoryl acceptor must remain oriented intracellularly, some ionophoric elements of the system must oscillate cis and trans with respect t o the phosphoryl acceptor and the plane of the membrane. Although the “cis-trans’’ concept adequately depicts the rudiments of active transport, it was not intended t o imply many of the attributes that are often associated with it. For example, it says nothing about the nature o r number of ionophoric elements associated with a phosphoryl acceptor. These elements could equally well be “leashed cages” or “everting funnels.” The model does imply that Na+ and K+ act sequentially, but it does not require that they interact with identical ionophoric elements. Many such conclusions are beyond the reach of enzyme kinetics.2i Moreover, certain relatively well established kinetic observations have not been rationalized in terms of the “cis-trans” model. We have attempted to deal with some of these, which include the kinetics of p-nitrophenylphosphate (NPP) hydrolysis. We will briefly review the evidence that has led us to postulate the participation of three distinct sets of monovalent cation activation sites in the hydrolysis of NPP by (Na+-K+)-ATPase. Following this we will present some studies of the interaction of nucleotides with these sites. An attempt will be made t o reconcile these observations of the NPPase system with the monovalent cation sites of the ATPase system. Finally, we will consider some species differences, possible alternative mechanisms, and the implications for active transport.

Electrophorus Adenosine Triphosphatase: Sodium-Activated Exchange after N-Ethyl Maleimide Treatment

The microsomal fraction from the electric organ of the eel Electrophorus electricus catalyzes the hydrolysis of adenosine triphosphate in the presence of Mg++, Na+, and K+. The same preparation catalyzes a Mg++-dependent transphosphorylation between adenosine triphosphate and adenosine diphosphate. Both of these reactions are inhibited after treatment of the microsomes with N-ethyl maleimide. However, the addition of Na+ reactivates the transphosphorylation, and the rate becomes more rapid than that of the original. This new Na+-sensitive exchange reaction is believed to be a component of the hydrolytic reaction.