A New Spectrophotometric Assay for Enzymes of Purine Metabolism

Abstract

A new spectrophotometric method for the determination of adenosine deaminase is described. Adenosine is deaminated to inosine, the latter is cleaved by an inosine-guanosine specific nucleoside phosphorylase to hypoxanthine and ribose-1-phosphate. Hypoxanthine can be oxidized further to uric acid by xanthine oxidase or to allantoin by xanthine oxidase and uricase. The hydrogen peroxide formed in these reactions is reduced by catalase to water. In the presence of high concentrations of ethanol, equivalent amounts of acetaldehyde are produced. The acetaldehyde is oxidized NAD(P) dependent and the production rate of NAD(P)H is recorded at 334 nm. The new method is suitable for the detection of adenosine deaminase in whole blood, lymphocytes, sera and tissues.

Vitamin D(3) contribution to the regulation of oxidative metabolism in the liver of diabetic mice

“…The activity of xanthine oxidase (EC 1.17.3.2) was determined in the reaction of ethanol oxidation by hydrogen peroxide (which was formed due to substrate hypoxanthine oxidation by xanthine oxidase) into acetaldehyde, which was cleaved by NAD-dependent aldehyde dehydrogenase. Reduction of coenzyme NAD + was detected with spectrophotometry (λ = 334 nm) [24].…”

Oxygen radicals as effectors of cartilage destruction. Direct degradative effect on matrix components and indirect action via activation of latent collagenase from polymorphonuclear leukocytes

“…The purified fraction contained no appreciable amounts of proteolytic activity, as assessed by the azocaseine assay, when 1 mg of XO was tested. The specific activity of the purified enzyme was determined by the method described by Heinz et al (19), and 1 mg was equivalent to an activity of 0.34 units.…”

Inosine: A bioactive metabolite with multimodal actions in human diseases

“…However, the PNP reaction equilibrium is biased toward inosine degradation due to the more significant concentration of inorganic phosphate than base and R-1-P ( Traut, 1994 ), and the linked reaction of hypoxanthine catalyzed by hypoxanthine phosphoribosyl transferase and xanthine oxidase ( Pugmire and Ealick, 2002 ; Il’icheva et al, 2020 ). Xanthine oxidase catabolizes hypoxanthine into uric acid via xanthine ( Heinz et al, 1980 ; Moriwaki et al, 1999 ; Doyle et al, 2018 ). Humans and higher primates excrete uric acid in their urine, but other mammals convert uric acid to allantoin by uricase and then excrete it in urine ( Heinz et al, 1980 ; Kurtz et al, 1986 ; Moriwaki et al, 1999 ).…”

“…Xanthine oxidase catabolizes hypoxanthine into uric acid via xanthine ( Heinz et al, 1980 ; Moriwaki et al, 1999 ; Doyle et al, 2018 ). Humans and higher primates excrete uric acid in their urine, but other mammals convert uric acid to allantoin by uricase and then excrete it in urine ( Heinz et al, 1980 ; Kurtz et al, 1986 ; Moriwaki et al, 1999 ). Uric acid, the end product of human purine metabolism, is one of the major antioxidants ( Ames et al, 1981 ; Whiteman et al, 2002 ; Muraoka and Miura, 2003 ) and protects against neurological and intestinal diseases ( Hooper et al, 1998 ; Hooper et al, 2000 ; Toncev et al, 2002 ; Ascherio et al, 2009 ; Matsuo et al, 2015 ; Yasutake et al, 2017 ).…”