A new assay for glutamate-oxaloacetate transaminase

✍ Scribed by Hajime Itoh; Paul A. Srere

• Publisher: Elsevier Science
• Year: 1970
• Tongue: English
• Weight: 294 KB
• Volume: 35
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(70)90202-2

✦ Synopsis

Glutamate-oxaloacetate transaminase (GOT) (n-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) is an extensively studied enzyme (1, 2) and its content in sera from a variety of patients has important clinical diagnostic value (3). A large number of methods are available for its determination

(1, 2). This enzyme-catalyzed reaction produces oxaloacetate, and it occurred to us that it could be measured by coupling it to the citrate synthase-DTNB method (4) for determining oxaloacetate. This paper presents such a method and shows that it is as rapid and convenient as the malate dehydrogenase coupled assay. In addition it offers the advantages of increased sensitivity, a visible change in color, so that spot tests may be run, and the ability of being used with inexpensive calorimeters. METHODS Enzymes and Chemicals Citrate synthase (EC 4.1.3.7)) malate dehydrogenase (EC 1.1.1.37), and glutamate-oxaloacetate transaminase were purchased from C. F. Boehringer and Soehne (Mannheim) . 5,5'-Dithiobis-(2nitrobenzoic acid) (DTNB) was obtained from Aldrich Chemical Co., Inc. (Milwaukee), CoA from P-L Biochemicals, Inc. (Milwaukee), pyridoxal phosphate and aspartic acid from Sigma Chemical Co. (St. Louis), cr-ketoglutarate from Calbiochem (Los Angeles), and citric acid from J. T. Baker Chemical Co. (Phillipsburg).

Acetyl-CoA was made with CoA and acetic anhydride according to the method of Simon and Shemin (5).

Measurement of GOT Enzyme Activity

Coupled citrate syntha.se (C'S) method. The complete reaction system contained in a final volume of 1.0 ml: 200 pmoles Tris-HCl, pH 8.1