Regional localization of the gene for human phosphoribosylpyrophosphate synthetase on the X chromosome.

Sixty-eight independent hybrid clones were isolated after irradiated normal human lymphocytes were fused with Chinese hamster fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase activity. The cells were grown under selective conditions requiring retention of the X chromosome-linked locus for human hypoxanthine-guanine phosphoribosyltransferase. The frequency and patterns of cotransference of human phosphoribosylpyrophosphate synthetase with the selected marker and with additional X-linked enzymatic markers confirm X linkage of the structural gene for human phosphoribosylpyrophosphate synthetase and support assignment of this gene to a position on the long arm of the X, between the loci for alpha-galactosidase and hypoxanthine-guanine phosphoribosyltransferase.

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates of incorporation of [14C]adenine and hypoxanthine into acid-soluble nucleotides during incubation at 1 mM Pi. These findings provided further confirmation of the extent to which PP-ribose-P synthesis is modulated in the normal cell at physiological Pi concentration by purine nucleotide inhibition of PP-ribose-P synthetase. The activity and kinetic characteristics of PP-ribose-P synthetase from fibroblasts of the mother of patient S.M. indicated that this woman was a heterozygous carrier of the enzyme defect expressed in hemizygous manner by her son.

Effects of 2'-deoxycoformycin on the metabolism of purines and the survival of malignant cells in a patient with T-cell leukemia.

The in vitro effects of deoxyadenosine and an adenosine deaminase inhibitor, deoxycoformycin, on the synthesis of DNA and the metabolism of purines were investigated in human leukemic T-cells. In the presence of 10 microM deoxycoformycin, the synthesis of DNA was completely inhibited by concentrations of deoxyadenosine of 10 microM or greater. In contrast, the synthesis of DNA in normal bone marrow cells was not inhibited in the presence of up to 20 microM deoxycoformycin and up to 10 microM deoxyadenosine. Following incubation of leukemia T-cells with deoxycoformycin and deoxyadenosine, there was a significant rise in the concentration of deoxyadenosine 5'-triphosphate which was accompanied by reductions in the concentrations of adenosine 5'-triphosphate and guanosine 5'-triphosphate, as revealed by high-pressure liquid chromatographic analysis. The effects of deoxycoformycin on T-cell leukemia were examined in vivo. A patient with acute T-cell leukemia in the terminal stage received five daily injections of 250 micrograms of deoxycoformycin per kg. Among the noted changes, most prominent was the drop in the leukocyte count. Initially, the cell count rose from 7,200 cells/microliters on Day 1 to 120,000 cells/microliters on Day 3. On Day 5, the cell count began to decline and reached a nadir of 600 cells/microliter on Day 10. The leukocyte count remained below 1,000 cells/microliter through Day 12. The reduction in cell count was preceded by a decline in the incorporation of [3H]thymidine in the cells, which dropped to negligible amount by Day 7. The other prominent change was a decrease in adenosine deaminase activity in both red cells and leukemic cells. Adenosine deaminase activity of red cells dropped to 5% on Day 4, and that of leukemic cells dropped to 59% on Day 5. In addition, there were considerable alterations in the concentrations of purine metabolites which were characterized by a progressive reduction in the concentrations of total purine metabolites, especially adenosine 5'-triphosphate, and a transient rise in the concentrations of deoxyadenosine 5'-triphosphate, adenosine 5'-monophosphate, and adenosine 5-diphosphate. These findings suggest that treatment with deoxycoformycin may be of therapeutic value for T-cell leukemia. It may provide opportunities for studying the purine metabolism in T-leukemic cells which could lead to better approaches to treatment.

Continuous monitoring of radioactivity of effluent from a high-speed amino acid analyzer, with a new system of sample segmentation.

Radioactivity in the effluents from high-speed liquid chromatographic analyzers was continuously measured by use of a new sample segmentation system. To achieve highest counting efficiency, the column effluents were mixed with scintillation fluid carrying small segments of semisolid, resillient spacer. After passing through a mixer, the mixture was passed through a hollow-tube flow cell mounted in a liquid scintillation spectrometer. Because the added spacer acts like a continuous series of pistons, it unifies flow rate at center and walls of the conduit and so decreases sample carryover. This system was applied to measurement of radioactivity in the effluent of a high-speed amino acid analyzer, in which some amino acids emerge at 30-s intervals. The resulting profiles were as well resolved as were the ninhydrin-color profiles. Counting efficiencies for tritium and carbon- 14 were comparable to those obtained by static counting in vials.

Activation of variants of hypoxanthine-guanine phosphoribosyl transferase by the normal enzyme.

Deficient hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) enzymes from erythrocytes of patients with hyperuricemia and with the Lesch-Nyhan syndrome migrate 15% faster in polyacrylamide gel disc electrophoresis than the normal enzyme. A half-sister of two males with partial deficiency, who had 34% of normal HGPRT activity in her erythrocytes, yielded profiles containing two distinct zones of activity; one corresponded to the enzyme found in normal individuals and one to the variant of her half-brothers. However, in her profile her variant enzyme showed notably greater activity than that observed in her half-brothers. This increase was due to an activation of the variant by normal enzyme. Electrophoresis of mixtures of normal enzyme with partially deficient enzymes from patients with hyperuricemia and with the Lesch-Nyhan syndrome also led to activation of deficient HGPRT variants by normal enzymes. Deficient variants were also activated by normal enzyme on filtration through Sephadex G-25. Experiments in which deficient variant enzymes were activated with purified normal enzyme labeled with (125)I indicated that deficient enzymes incorporate components of the normal enzyme. No such activation of deficient enzymes was ever obtained when mixtures of deficient and normal enzymes were put together in a test tube.

Human GMP synthetase.

The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes, placenta, and liver. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration. The Km values were determined to be 4.9 microM for XMP, 270 microM for ATP, and 340 microM for glutamine. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient. The enzyme was specific for ATP as the energy source. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

5'-Nucleotidase: solubilization, radiochemical analysis, and electrophoresis.

5'-Nucleotidase (5'-NT, E.C. 3.1.3.5) of cultured human and rodent cells was rendered soluble using the zwitterionic detergent Zwittergent 314. Optimal activity of 5'-NT was obtained when sonicated cells were incubated in solutions containing 0.75% (w/v) Zwittergent. A method was developed for the determination of the activity of 5'-NT in which the unutilized substrate, [14C]-AMP, was precipitated with lanthanum chloride and the soluble [14C]-adenosine was measured by scintillation counting. 5'-NT isozymes were separated using agarose gel electrophoresis and isoelectric focusing in polyacrylamide gel. The zones of enzyme activity were established by precipitation of unutilized [14C]-AMP with LaCl3, removal of soluble [14C)-adenosine by washing gels in water, and autoradiography. The zones of 5'-NT appeared as clear zones on darkened X-ray film. When analyzed by agarose gel electrophoresis, fibroblasts derived from human skin and rat liver produced a single zone of 5'-NT activity. The 5'-NT isozyme of rat cells migrated faster than that of human cells and was easy to distinguish. The presence of detergent in the sample and in the gel enhanced enzymatic activity and improved the separation of the isozymes. Isoelectric focusing resolved 5'-NT of human fibroblasts into two molecular forms, one of which focused in the region of pH 6 and the other at pH5.