Glutamine transaminase K and cysteine S-conjugate β-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-phenylalanine, α-keto-γ-methiolbutyrate (αKMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and αKMB. 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 β-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing αKMB (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 β-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 ∼ 95,000). This band coincides with both the cysteine S-conjugate β-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 β-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K. Activity staining of rat kidney homogenates (and to a lesser extent rat liver homogenates) for glutamine transaminase K and cysteine S-conjugate β-lyase activities revealed the presence of an additional band. The enzyme responsible for the second band of activity was estimated to have an apparent molecular weight of ∼ 330,000. The ratio of β-lyase to transaminase activity was greater for the higher molecular weight form than for the lower molecular weight form.