Abstract
This chapter describes the purification procedure of guanylate kinases from human erythrocytes, hog brain, and rat liver. A convenient assay involves the measurement of both adenosine diphosphate (ADP) and guanosine diphosphate (GDP) formed from adenosine triphosphate (ATP) and guanosine monophosphate (GMP) to pyruvate kinase and lactate dehydrogenase reactions. The decrease in absorbancy at 340 nm associated with NADH oxidation is measured with a spectrophotometer using a blue filter. All procedures steps are performed at 0–4°. Enzymic activities and protein concentrations are determined after each step of purification. The purified guanylate kinases from the sources that are given in the chapter, were found stable for several years when stored below 0° in the presence of 70% saturated ammonium sulfate. In experiments with partially purified erythrocytic guanylate kinase, significant activity was retained for several minutes after precipitation at 3% perchloric acid followed by neutralization with potassium hydroxide.
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