A New Spectrophotometric Method for the Determination of 5′-Nucleotidase

Abstract

Summary: A Spectrophotometric method is described for the determination of 5'-nucleotidase.In combination with the enzymes nucleoside phosphorylase and xanthine oxidase, inosine, formed by hydrolysis of S'-IMP by S'-nucleotidase, is cleaved phosphorolytically to hypoxanthine, which is oxidized to uric acid. In the presence of ethanol, the hydrogen peroxide formed is reduced by catalase and equivalent amounts of acetaldehyde are produced. The aldehyde is dehydrogenated (NADP-dependent) by aldehyde dehydrogenase and the production ι rate of NADPH is recorded at 334 nm. The inhibition of the unspecific cleavage of 5'-IMP by phosphatases is examined critically. Eine neue spektrophotometrische Methode zur Bestimmung von 5'-Nucleotidase lZusammenfassung: Eine spektrophotometrische Methode f r die Bestimmung der S'-Nucleotidaseaktivit t wird be-! schrieben. ;In Kombination mit den Enzymen Nucleosidphosphorylase und Xanthinoxidase wird Inosin, das bei der Hydrolyse i von S'-IMP durch die S'-Nucleotidase entsteht, phosphorolytisch zu Hypoxanthin gespalten, das weiter zu Harns ure j oxidiert wird. Das entstandene Wasserstoffperoxid wird mit Katalase in Gegenwart von Ethanol reduziert. Dabei ent-! stehen quimol fe Mengen an Acetaldehyd. Der Aldehyd wird NADP-abh ngig durch die Aldehyddehydrogenase l dehydriert und das gebildete NADPH bei 334 nm automatisch registriert. Die Inhibierung der unspezifischen S'-IMP-} Hydrolyse wird kritisch untersucht.

Continuous Assay of Serum 5'-Nucleotidase Activity with Inosine 5'-Monophosphate as Substrate and Total Automation Using a Transfer-Analyzer (Kem-O-Mat)

“…The principle used here for measuring serum 5'-nucleotidase catalytic activity was originally proposed by Dooley & Racich and Heinz et al (12) and reproduces in vitro the scheine of purine nucleotide degradation in vivo, up to the uric acid stage, the final…”

“…Of the two procedures involving the use inosine 5'-monophosphate (11-12), we thought it preferable to choose that of Heinz et al (12), which measures the H 2 O2 formed, a product which, unlike uric acid, is not initially present in the serum.…”

“…Adaptation to a Kem-O-Mat transfer analyzer of the principle of measurement suggested by Heinz et al (12) enabled us to redefine some of the method's parameters.…”

“…Principle [8][9][10][11][12][13][14] C]Inosine S'-monophosph te is choseri s Substrate; it is hydrolysed by 5'*nucleotidase and non-ŝ pecific alkaline phosphatases to yield [8^1 4 C]inosine and inorganic phosphate. Glycerol 2-phosphate acts s "divefsion Substrate", it is preferehtially hydrolysed by non-specific alkaline phosphatases.…”

Purification and characterization of xanthine oxidase from liver of the water buffalo Bubalus bubalis

“…XO has a quaternary structure in which each subunit contains molybdopterin, flavin adenine dinucleotide and ironsulfur center (Kuwabara et al, 2003). This enzyme is widely used as a detector for nucleotidases, purines, superoxide dismutases, adenosine deaminase, phosphates of blood serum and in the determination of liver diseases (Heinz et al, 1980;Groot and Noll, 1985). It had also been reported as playing vital roles in innate immune system (Vorbach et al, 2003), diseases of cardiovascular system (Berry and Hare, 2004) and employed as antimicrobial agent (Martin et al, 2004, Zhang et al, 2012.…”