A Simplified Procedure for the Assay of 5’-Nucleotidase

5’-Nucleotidase activity is measuredby inhibiting phosphataseactivity with 0.01 M Ni+2 and eliminating the requirement for protein removal before measurement of phosphate. The normal range for adults is given as 1.6-14.0 units/liter of serum, with no apparent difference between the sexes. “T”HE NEED for differentiating the origin of the alkaline phospliatases in serum as being primarily bone or liver has prompted the use of the assay for 5’-adenylic acid (5’-AMP) phosphatase * (commonly referred to as 5’-nucleotidase) as a measure of heptobiliary function. The role of this enzyme in the mentioned capacity has been evaluated con-siderably with generally acceptable results (1-9). Many technics have been offered for assaying this enzyme (2, 3, 5, 8, 10), and those em-ploymg inhibition by nickel ion have appeared to function best. This latter procedure, which uses as a measure of 5’-nucleotidase activity the inhibition of phosphatase activity by 0.01 NI Ni2, requires the measurement of inorganic phosphorus. A drawback to the use of this technic is the necessity for precipitating the protein prior to analysis. In an effort to overcome this, a simple procedure which eliminates the need for removal of the protein was developed ill this laboratory and the procedure is reported herein. The assay is similar in principle to that of Campbell (10) and the protocol is as follows: Tube A: 0.1 ml serum; 2 ml of a mixture con-taining 5’-AMP (0.002 NI), sodium barbital (0.02 NI, pH 7.5), and MgCI2 (0.04 NI). Tube B contains all of the above solutions pills 0.02 ml of 1 NI NiCL,. Both tubes are incubated at 37#{176}for 1 hr. The reaction is terminated by adding to both tubes 1.0 ml of a 4 % (w/v) sodium lauryl sulfate (SDS; Mann Research Labs) and 0.5 ml of 4 NI sodium acetate buffer, pH 4.5. To Tube A is added 0.02 ml of 1 NI NiCl2 to compensate for any phosphate present in this reagent. Then 0.2 ml o