Monoclonal antibodies against rat brain hexokinase. Effects on catalytic function and binding to the outer mitochondrial membrane.
A library of seven monoclonal antibodies has been prepared against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Four of these antibodies, all of the IgG class, react with both native hexokinase and, to a more limited extent, with a ureadenatured S-carboxymethylated form of the enzyme. The other three antibodies, all of the IgM class, react readily with the denatured enzyme but are quite ineffective at binding native hexokinase. The monoclonal IgGs were further characterized with respect to their effects on certain functional properties of hexokinase. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. One of the antibodies, designated 3C, totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme in either the absence or presence of glucose 6-phosphate, a ligand which promotes solubilization of mitochondrial hexokinase. It was concluded that the epitope recognized by 3C lies in, or immediately adjacent to, the region of the hexokinase molecule directly involved in interaction with the mitochondrial membrane. Two other IgGs, designated 1B and 2B, had only marginal effects on the binding of hexokinase to mitochondria, but were highly effective in preventing solubilization of the mitochondrially bound hexokinase by glucose 6-phosphate. Since these antibodies did not prevent binding of this ligand, as evidenced by the lack of an effect on inhibition, it is suggested that the effect of 1B and 2B on glucose 6-phosphate-induced solubilization is due to selective modification of the conformational changes that result from binding of glucose 6-phosphate. The monoclonal IgG designated 13 had no appreciable effect on either binding or glucose 6-phosphate-induced solubilization. The epitope for 13 is thought to lie in a "neutral" region of the hexokinase molecule, not involved in either catalytic or membrane-binding functions of the enzyme.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001921:Brain / Q000201:enzymology*; D006593:Hexokinase / Q000276:immunology / Q000378:metabolism*; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D006593:Hexokinase