Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.
A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005972:Glutaminase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D017830:Octoxynol; D010446:Peptide Fragments / Q000032:analysis; D010710:Phosphates / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D010446:Peptide Fragments; D010710:Phosphates; D011092:Polyethylene Glycols; D017830:Octoxynol; D005972:Glutaminase