Rat lens glutaminase: separation and characterization of soluble and particulate fractions.
The ability of rat lenses to import glutamate from the aqueous humor is limited by the activity of the transporter for dicarboxylic acids in this tissue. The principal source of glutamate for rat lens metabolism appears to be glutamine, which enters the lens much more readily than glutamate and is then deamidated by glutaminase. Both soluble and particulate glutaminase activities were obtained from rat lenses by extraction and differential centrifugation. The lens fractions were compared with previously reported isoenzymes of glutaminase from rat kidney and liver. The apparent Km for glutamine of the soluble preparation from lens was in the range from 17- to 24 mM, while that of the particulate lens fraction was 5 mM. Gel filtration on Sepharose 4B demonstrated that the lens soluble glutaminase in Tris buffer is similar in size to the kidney enzyme and, like the glutaminase from kidney, reversibly formed active aggregates in borate buffer. The liver enzyme did not form aggregates under identical conditions. The glutaminase preparations were all dependent on phosphate for complete activation, but the lens particulate fraction is more active than the lens or kidney soluble fractions at low concentrations of inorganic phosphate. Unlike the soluble enzyme, the particulate glutaminase was partially activated by phosphorylcholine. Rat lenses contain 11 mM phosphorylcholine, and this unusually high concentration of phosphorylcholine may be sufficient to partially activate the enzyme. These properties of the particulate fraction suggest that the membrane-bound glutaminase may be physiologically important in the lens in vivo.
Vallari
A S
AS
Department of Biochemistry, University of Tennessee, Memphis 38163.
Macleod
R M
RM
Jernigan
H M
HM
D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004789:Enzyme Activation; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008297:Male; D010710:Phosphates / Q000378:metabolism; D010767:Phosphorylcholine / Q000494:pharmacology; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D007527:Isoenzymes; D010710:Phosphates; D010767:Phosphorylcholine; D020245:p-Chloromercuribenzoic Acid; D005972:Glutaminase