Determination of Serum Nucleotidase with Cytidine Monophosphate as Substrate. Part II: Improvement of the procedure

Determination of Serum Nucleotidase with Cytidine Monophosphate as Substrate. Part II:... Introduction Recently, we described a new coupled colorimetric assay for serum activity toward cytidine-S'-monophosphate (5'-nucleotidase activity, EC 3.1.3.5) based on measurement of the equivalent amount of ammonia released from cytidine by cytidine deaminase (EC 3.5.4.5) (1). In developing this assay we used a crude cy tidinedeaminase preparation as the auxiliary enzyme, which resulted in rather high background extinction values and an overcorrection with respect to the reagentcontrol, probably due to an enzyme contaminating the cytidiiie deaminase. To circumvent this problem, we offered an indirect method. In this communication we prove that a simple and rapid purification of the cytidine deaminase is sufficient to give a reliable direct method, with none of the above disadvantages. Materials and Methods Reagents 1. Buffer solution: Dissolve 4.20 g sodium diethylbarbiturate and 6.30 g MgSO4 · 7H2O in about 800 ml of distilled water. Adjust the pH to 7.50 with HC1 (1 mol/1) and dilute to 1,000 ml. 2. Cytidine deaminase solution: Dilute the stock cytidine deaminase (see below) with buffer solution to give an activity of about 225 U/l and dissolve J. Clin. Chem. Clin. Biochem. / Vol. 14,1976 / No. 10 28 mg disodium phenylorthophosphate (British Drug Houses) per 10 ml http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Chemistry and Laboratory Medicine de Gruyter

Determination of Serum Nucleotidase with Cytidine Monophosphate as Substrate. Part II: Improvement of the procedure

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Publisher
de Gruyter
Copyright
Copyright © 2009 Walter de Gruyter
ISSN
1434-6621
eISSN
1437-4331
DOI
10.1515/cclm.1976.14.1-12.469
Publisher site
See Article on Publisher Site

Abstract

Introduction Recently, we described a new coupled colorimetric assay for serum activity toward cytidine-S'-monophosphate (5'-nucleotidase activity, EC 3.1.3.5) based on measurement of the equivalent amount of ammonia released from cytidine by cytidine deaminase (EC 3.5.4.5) (1). In developing this assay we used a crude cy tidinedeaminase preparation as the auxiliary enzyme, which resulted in rather high background extinction values and an overcorrection with respect to the reagentcontrol, probably due to an enzyme contaminating the cytidiiie deaminase. To circumvent this problem, we offered an indirect method. In this communication we prove that a simple and rapid purification of the cytidine deaminase is sufficient to give a reliable direct method, with none of the above disadvantages. Materials and Methods Reagents 1. Buffer solution: Dissolve 4.20 g sodium diethylbarbiturate and 6.30 g MgSO4 · 7H2O in about 800 ml of distilled water. Adjust the pH to 7.50 with HC1 (1 mol/1) and dilute to 1,000 ml. 2. Cytidine deaminase solution: Dilute the stock cytidine deaminase (see below) with buffer solution to give an activity of about 225 U/l and dissolve J. Clin. Chem. Clin. Biochem. / Vol. 14,1976 / No. 10 28 mg disodium phenylorthophosphate (British Drug Houses) per 10 ml

Journal

Clinical Chemistry and Laboratory Medicinede Gruyter

Published: Jan 1, 1976

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