BIOCHEMICAL

The

MEDICINE

Formation

(1975) 13, 178-183

of SPhosphori in Human

leukemic

bosyl-1

-Pyrophosphate

Leukocytes

TOMIHIKO HIGUCHI, TORU NAKAMURA, AND GYOICHI WAKISAKA The First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University. Kyoto, Japan Received April 16, 197.5

INTRODUCTION Synthesis of purine nucleotides from preformed purines in the cells has been shown to be controlled more extensively by the endogenous supply of phosphoribosylpyrophosphate (PRPP) and rather less by the activity of the pruine phosphoribosyltransferase in experimental animal tumor cells (1)) human erythrocytes (2), and lymphocytes (3). We have also observed high hypoxanthine guanine phosphoribosyltransferase (HGPRTase) activity in human leukemic leukocytes from the patients with various types of leukemia (4). But there is no information regarding the supply of PRPP in human leukemic leukocytes. It is of considerable interest to clarify the supply of PRPP to understand the mechanism responsible for the conversion of purine bases to their nucelotides in human leukemic leukocytes. This report deals with the concentration of PRPP and the activity of the PRPP formation in human leukemic leukocytes, and offers some discussions concerning the mechanisms responsible for the supply of PRPP. MATERIALS AND METHODS All the methods used for preparing cell suspension of human erythrocytes, human leukemic leukocytes, and L1210 cells have been described in our previous publication (4). Human leukemic leukocytes (1 x 10’) were suspended in 1 ml of 0.9% NaCl containing 10 mM glucose and 60 mM inorganic phosphate. The suspensions were incubated for 60 minutes at 37” and the PRPP concentrations were determined as described below. Extraction of PRPP. After the incubation of the cell suspension, the leukocytes of L1210 cells were sedimented by centrifugation by 600 g for 10 minutes. The sedimented cells were resuspended in 1 mM EDTA, pH 7.4, at a concentration of 2 X lo7 cells/ml. The suspensions were heated by placing the test tubes in boiling water for 2 minutes and then promptly chilled in an ice bath. The denatured protein was removed by centrifuga178 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

PmP

FORMATION

AND

CONCENTRATION

179

tion and then the clear supernatant fluid was subjected immediately for assay of the PRPP contents. The recovery test showed that the presence of 1 mM EDTA completely prevented the decomposition of PRPP during the 2-minute heating for the extraction. Assay ufPRPP contents. The PRPP content of leukocytes or L1210 cells was determined by a slight modification of the procedure previously described by Henderson and Khoo (5). The PRPP content of the cell extracts was assayed by measuring the amounts of inosinic acid (IMP) formed in the reaction: hypoxanthine + PRPP -+ IMP + PPi, where [8J4C]hypoxanthine (20.7 mCi, 1 mM) and HGPRTase were in excess. The reaction mixture contained 55 pmoles Tris buffer, pH 7.4, 5 ,umoles MgCl*, 10 nmoles [8J4C]hypoxanthine, 0.09 ml of the cell extracts and 0.1 ml of hemolysates in a total volume of 0.25 ml. After 20 minutes incubation at 37”, the reaction was stopped by addition of 2 pmoles of neutralized EDTA, and an aliquot (20 ~1) of the reaction mixture was spotted on Toyo No. 51 paper with appropriate carriers. IMP produced was separated from the substrate by a high-voltage electrophoresis using 0.05 M borate buffer, pH 9.0, containing 0.001 M EDTA at 4000 V for 20 minutes (6). The chromatographic paper was dried in air and cut into 0.5-cm strips after working position of added carriers under a uv lamp. The radioactivities were counted in a Packard Tri-Carb scintillation counter. The amounts of PRPP in the assay medium were calculated from the IMP produced. The standard curve showed a linear responsiveness up to 5 pmoles of PRPP. Enzyme preparation. Tw.o times ten to the ninth packed erythrocytes were hemolyzed by adding 1 ml of 0.2 M Tris buffer, pH 7.4, and the hemolysates were used as crude HGPRTase for the assay of PRPP. RESULTS

The amounts of PRPP in heat extracts of human leukocytes were assayed as described in Materials and Methods. Using this radiochemical assay, it is possible to detect the amounts of PRPP as low as 0.03 nmoles/3 x 10’ cells in leukocytes. However, no significant radioactivity was detected in the nucleotide region of the chromatogram under these conditions in any of leukocytes examined. In contrast to human leukemic leukocytes, considerable amounts of PRPP (4.9 nmoles/3 x lo7 cells) were found in L1210 cells. This value agrees fairly well with the results reported by Henderson and Khoo (5) (0.28 pmoles/g in Ehrlich ascites carcinoma cells) and Paterson and Wang (7) (0.18 pmoles/g in Ehrlich ascites carcinoma cells) (Table 1). As shown in Fig. 1 leukocyte suspension from normal controls formed PRPP at the similar rates, which fluctuated within a narrow range. In sharp contrast to normal controls, leukocytes from the patients with

180

HIGUCHI,

NAKAMURA, TABLE

PRPP

AND

WAKISAKA

1

CONCENTRATION OF HUMAN LEUKEMIC REACTION CONDITIONS WERE THE SAME MATERIALS AND METHODS

LEUKOCYTES. AS THOSE

THE IN

PRPP concentration”

Cells

4.9

L1210 cells Leukemic leukocytes SY 28 F AML KD 36F AML YK 56M AMoL

The formation of 5-phosphoribosyl-1-pyrophosphate in human leukemic leukocytes.

BIOCHEMICAL The MEDICINE Formation (1975) 13, 178-183 of SPhosphori in Human leukemic bosyl-1 -Pyrophosphate Leukocytes TOMIHIKO HIGUCHI, TO...
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