Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.
An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)
Abraham
D G
DG
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Cooper
A J
AJ
D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology; D008190:Lyases / Q000032:analysis* / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D000637:Transaminases / Q000032:analysis* / Q000378:metabolism
D009580:Nitroblue Tetrazolium; D010649:Phenylalanine; C014632:2-keto-4-methylthiobutyric acid; C039961:S-(1,2-dichlorovinyl)cysteine; D008715:Methionine; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases; D003545:Cysteine