title: Properties of rat renal phosphate-dependent glutaminase coupled to Sepharose. Evidence that dimerization is essential for activation.
abstract: In the absence of phosphate, purified rat renal phosphate-dependent glutaminase exists as a catalytically inactive protomer. The addition of phosphate results in both dimerization and activation of the glutaminase. Covalent attachment of the dimeric form of the glutaminase to CNBr-activated Sepharose was achieved with 84% retention of activity. At least 70% of the bound glutaminase activity was expressed even in the absence of added phosphate. In addition, 6-diazo-5-oxo-L-norleucine, which interacts only with the catalytically active form of the glutaminase, inactivates the bound dimeric form of glutaminase at the same rate in either the absence or the presence of added phosphate. Therefore retention of dimeric structure is apparently sufficient to maintain glutaminase activity. In contrast, the coupling of the protomeric form of the enzyme to Sepharose resulted in retention of only 3% of the phosphate-induced glutaminase activity. However, up to 48% of this activity could be reconstituted by addition of soluble glutaminase under conditions that promote dimerization. These results indicate that the monomeric form of the glutaminase has minimal inherent activity and that dimerization is an essential step in the phosphate-induced activation of the glutaminase.
mesh_terms: D000818:Animals; D003980:Diazooxonorleucine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008744:Methylamines / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology*; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D012685:Sepharose / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D008744:Methylamines; D010710:Phosphates; D011134:Polysaccharides; D003980:Diazooxonorleucine; D012685:Sepharose; D005972:Glutaminase