Rat hepatic glutaminase: purification and immunochemical characterization.
A method for the purification of phosphate-activated glutaminase from the liver of streptozotocin-diabetic rats is described. The procedure involves solubilization of glutaminase activity from isolated mitochondria by sonication, followed by ammonium sulfate precipitation, polyethylene glycol precipitation, and sequential chromatography on DEAE, hydroxylapatite, and zinc-chelated resins. The enzyme was purified 600-fold to a specific activity of 31-57 U/mg protein. The purified enzyme has an apparent subunit molecular mass of 58,000-Da and is greater than 80% pure by scanning densitometry of sodium dodecyl sulfate-polyacrylamide gels. The purified enzyme has an apparent Km for glutamine of 17 mM and a pH optimum between 7.8 and 8.2. The physical and kinetic properties of this enzyme are similar to those of the enzyme from normal rat liver. Polyclonal antibodies raised against the enzyme specifically inhibit hepatic glutaminase activity and react primarily with a 58,000-Da peptide in liver fractions on immunoblots. These antibodies were used in equivalence point titrations and immunoblots to provide evidence for increased concentration of glutaminase protein in the liver of diabetic rats with no change in specific activity of the enzyme. In addition, the antibodies cross-react, at low affinity, with kidney-type glutaminases. On immunoblots, the antibodies did not react with fetal liver, mammary gland, or lung. Antibodies to rat hepatic glutaminase should prove useful as tools to study the long-term regulation of the enzyme.
Smith
E M
EM
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
Watford
M
M
D000645:Ammonium Sulfate; D000818:Animals; D002498:Centrifugation; D011232:Chemical Precipitation; D002845:Chromatography; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D005972:Glutaminase / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007163:Immunosorbent Techniques; D007421:Intestine, Small / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D005972:Glutaminase; D000645:Ammonium Sulfate