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Inhibition of Tritiated Thymidine Incorporation into DNA by Alkaline Phosphatase Preparations^*

Joseph R. Rubini, Sue Keller and Mary Sue McCall

( Veterans Administration Hospital and the University of Texas Southwestern Medical School, Dallas, Texas)

A factor present in homogenized normal human blood leukocytes containing alkaline phosphatase activity was shown to be effective in depressing the incorporation of TDR-H³ into DNA-H³ when preincubated with dog bone marrow, lymph, and human chronic granulocytic leukemia cell suspensions. Human donor blood buffy coats were extracted, and an approximately 200-fold purified preparation of leukocyte alkaline phosphatase was prepared by the method of Traubowitz. The purified leukocyte alkaline phosphatase preparation readily dephosphorylated thymidylic acid and was free of detectable thymidine phosphorylase activity. This purified leukocyte enzyme preparation was also seen to be active in depressing the incorporation of TDR-H³ into DNA-H³ when preincubated with the intact proliferative test cells. The active factor present in the purified alkaline phosphatase preparation was heat-labile, nondialyzable, and appeared to be inhibited by 10^-3 M Zn ion—all properties shared by alkaline phosphatase. It is suggested that the identity of this inhibiting factor is the enzyme alkaline phosphatase and that entrance of this enzyme into proliferative cells occurred via pinocytosis. A mechanism of action of alkaline phosphatase is proposed in which its capacity to dephosphorylate thymidylic acid monophosphate to thymidine could divert significant amounts of this essential deoxynucleotide from DNA formation. This diverting effect of alkaline phosphatase on thymidylate may play an important role in controlling DNA synthesis and growth.

^* Supported in part by U.S.P.H.S. Grant No. C 5325 and Atomic Energy Commission Contract No. AT (40-1) 2731.

Present address: Hematology Section, Veterans Administration Hospital, Coral Gables, and University of Miami School of Medicine, Miami, Florida.

Received 9/25/63.