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A Comparative Biochemical Study of Alkaline Phosphatases in Normal and Leukemic Mice¹

Department of Biophysics [H. N., K. J. W., R. H.-G.], and Department of Experimental Biology [N. H-G.], The Weizmann Institute of Science, Rehovot, Israel

Optimal conditions for maximal extractions of alkaline phosphatase (APase) present in the thymuses, spleens, livers, and kidneys of C57BL/6, SJL/J, and AKR strains of mice, leukemic and nonleukemic, were selected. Standard assay conditions for measuring APase activities were found by studying the dependencies of the observed enzymatic activities on pH, concentrations of MgCl₂, NaCl, and Tris. Mg⁺⁺ ions had inhibitory effects on the measured APase activities of the homogenates of all tissues to a varying extent; therefore Mg⁺⁺ was omitted from all the standard assays. NaCl concentrations up to 2.0 M only slightly affected the observed APase activity. On the basis of this observation, the pH dependence of APase activity could be measured without adjusting the ionic strength at each pH. Differences were noted in the pH profiles of APase's of the thymus and kidney as compared to those of liver and spleen. Elevated APase activity was noted with increasing Tris concentration (up to 0.5 M), without a pattern specific to either organ, strain, or normal and leukemic mice.

The most significant difference between the APase's derived from leukemic and nonleukemic tissue homogenates was observed when the rates of hydrolyses of p-nitrophenyl phosphate and cysteamine S-phosphate were compared. In all leukemia tissue homogenates, the ratio of the rate hydrolysis of p-nitrophenyl phosphate to the rate of hydrolysis of cysteamine S-phosphate was found to be greater than the corresponding ratio for the nonleukemic tissue homogenate. An enhanced thermal instability of APase derived from leukemic sources was observed compared to that from nonleukemic sources during preincubation at 55° followed by assay of the remaining enzymatic activity. No differences could be noted between the APase's of the different tissues, either leukemic or nonleukemic, when L-phenylalanine inhibition was measured.

In the case of the AKR strain, a difference in the relative mobility rates on acrylamide gels was noted in the APase isoenzyme pattern from the thymus between leukemic and nonleukemic donors. In other cases, only minor differences in the relative mobility rates were observed between tissues from different strains and between tissues within the same strain.

¹ This work was supported by a grant from the Israel Cancer Association.

² Present address: Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, Zürich, Switzerland.

Received 2/22/71. Accepted 7/ 6/71.

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