M. Earl Balis

Rarity of X-Linked Partial Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency in a Large Gouty Population

Abstract In a survey of 425 cases of hyperuricemia with gouty arthritis or uric acid stone, or both, we found partial deficiency of H-G PRTase, a newly recognized cause of these manifestations, in ...

Cell population density and phenotypic expression of tissue culture fibroblasts from heterozygotes of Lesch-Nyhan's disease (inosinate pyrophosphorylase deficiency)

Radioautographic examination of skin fibroblasts grown in tissue culture from normal donors revealed heavy labeling of almost all cells following incubation with tritiated hypoxanthine. Cells from patients with Lesch-Nyhans disease, lacking inosinate pyrophosphorylase, had only 10 grains or less per cell. When normal and abnormal cells were mixed prior to culture, there was a progressive increase, with culture time, in the percentage of heavily labeled cells so that by 96 hr, when the cells were confluent, over 95% of the cells were heavily labeled. Reduction of cell density by subculture produced a reversion to original values. Cultures from three obligatory heterozygotes revealed the expected mixed population of cells. This appears to be a practical approach to the identification of the heterozygote.

Glutaminase activity of L-asparagine amidohydrolase☆

Relatively high concentrations of 6-diazo-5-oxo-norleucine (DON) and azaserine, potent specific inhibitors of many enzymes using glutamine as substrate, do not have an appreciable effect on the activity of Escherechia coliL-asparagine amido-hydrolase (EC 3.5.1.1) when either asparagine or glutamine is used as substrate. Glutamine acts as a competitive inhibitor when asparagine hydrolysis is measured, and asparagine inhibits glutamine hydrolysis. These observations, together with the fact that no one has been able to separate these two activities of the E. coli enzyme, point strongly to the presence of a single enzymatic site. However, the two activities have different pH dependencies. The glutaminase activity shows a 6-fold increase from pH 5 to pH 8·7; the asparaginase activity is almost constant over this pH range. n nThe measured Michaelis constants and maximum velocities for the glutaminase and asparaginase activities at pH 8·7 are as follows KmG = 1·3 × 10−2M; KwA = 1·3 × 10−3M;VmaxG = 0·080 μmoles pei min per i.u. of asparaginase activity; VmaxA = 1·1 μmoles per min per i.u.The inhibition dissociation constant for glutamine acting as inhibitor of asparaginase in 1·2 × 10−2.

The effects of 6-mercaptopurine on Lactobacillus casei

Abstract The inhibitory action of 6-mercaptopurine has been shown to be manifested by an interference with purine interconversion. The incorporation of adenine and of adenylic acid are affected in different ways by 6-mercaptopurine. These facts have been fitted to a schematic representation of a purine interconversion cycle compatible with the known metabolic behavior of L. casei.

Nucleic acid metabolism in the gastrointestinal tract of the mouse during fasting and restraint-stress.

Abstract The uptake of thymidine-methyl- 3 H and uridine-5- 3 H into DNA and RNA of gastrointestinal tissues of mice has been measured after 16 and 40 hours of fasting and restraint. Fasting had little effect on DNA or RNA metabolism in the gastrointestinal tract except for increases in the uptake of uridine in the small intestine. These increases are thought to represent changes in uridine pool size. Restraint caused a reduction in DNA synthesis along the entire gastrointestinal tract and affected RNA metabolism in the stomach but not in the small intestine or colon. The changes in the stomach included early loss of total RNA followed by a decrease in the uptake of uridine-5- 3 H. The latter appeared to coincide wit the occurrence of gastric erosions. The importance of RNA metabolism in mucus production and in the maintenance of the mucosal cell population has been discussed in relation to the development of stress-induced gastric erosions.

Investigations on amino acids bound to DNA

Abstract A small but persistent amount (0.1–0.6 %) of amino acids is found bound to the DNA of all tissues examined. The amount is constant and reproducible for the DNA of a given tissue taken from animals of the same species, sex and age. These observations suggest that the residues have a role. There is a difference in absolute and relative amounts of the amino acids bound to DNA from different tissues of the same species. There are also differences among the amino acids bound to the DNA of the liver of several species.

Inosinate pyrophosphorylase activity in immature blood cells in X-linked congenital hyperuricosuria

Radioautographic examination of the bone marrow of three patients with X-linked congenital hyperuricosuria (Lesch-Nyhans disease), after incubation with radioactive hypoxanthine, revealed no labeling even in extremely immature blood cells. An adequacy of the cosubstrate, phosphoribosepyrophosphate, in these cells was demonstrated by the appearance of labeling following incubation with tritiated adenine. It is concluded that the enzyme IMP pyrophosphorylase (inosinate: pyrophosphate phosphoribosyltransferase E.C.2.4.2.8) is deficient in young blood cells as well as in peripheral blood elements. The possibility that the enzyme deficiency in the peripheral blood results from the rapid degradation of an unstable enzyme is thereby excluded. Absence of radioactive labeling of terminal cells—erythrocytes, granulocytes, and platelets—is attributed to an insufficiency of the cosubstrate, phosphoribosepyrophosphate.

Influence of age on glycine and purine metabolism.

Abstract Incorporation of adenine and glycine into ribonucleic acid adenine of young and old hamsters has been determined. There is apparently a greater utilization of exogenous precursors of RNA purines in old than in young hamsters. In actuality this is due to differences in endogenous precursor metabolism. The pool with which administered adenine is immediately mixed is about 0.6 mmole/kg. of body weight in mature animals; it varies from tissue to tissue in weanlings. The nonprotein glycine of both adult and young hamsters is about the same at any instant. However, the glycine pool of the young animals seems to be much more rapidly renewed, and the total free glycine available in young hamsters is greater than that in old. The apparent low incorporation of glycine into the nucleic acid adenine of young animals can be explained on the basis of these observations.

Stability of AMP: Pyrophosphate phosphoribosyltransferase, an autosomally determined enzyme in an X-linked disease identification of a destabilizer

Abstract The stability of AMP: pyrophosphate phosphoribosyltransferases (AMP pyrophosphorylase) from erythrocytes of normal subjects and patients with Lesch-Nyhan disease has been studied. Storage of intact cells, but not lysates, led to increased heat stability of the normal but not the Lesch-Nyhan enzyme. Passage of lysates of stored normal erythrocytes through Sephadex gave a further increase in the heat stability of AMP pyrophosphorylase. Boiled extracts of stored erythrocytes contained a potent destabilizer of the enzyme which was identified as hypoxanthine. A heat stable form of AMP pyrophosphorylase was generated by pre-incubation with 5-phosphoribosyl i -pyrophosphate, a substrate. These observations suggest that the level of AMP pyrophosphorylase in the erythrocyte may be controlled by the relative concentrations of stabilizer and destabilizer. A variety of purine derivatives were destabilizers of the enzyme. Compounds with 6-amino substituents were active with the normal and Lesch-Nyhan enzyme but 6-hydroxypurines affected only normal AMP pyrophosphorylase. AMP pyrophosphorylases were purified 200-fold from normal and Lesch-Nyhan erythrocytes. The purified enzymes retained some of the differences in stability seen with crude lysates, but the destabilizing effect of purines was lost.

EFFECT OF AMINO ACIDS ON THYMINE DIMERIZATION

Abstract— The effect of several amines on the U.V. catalyzed dimerization of thymine was determined at 25† and — 20†. Most amino acids and EDTA had no effect. Histidine, phosphoserine and phosphoethanolamine increased the net yield of dimer at 25†, but decreased the yield at — 20†. Histidine stimulates the cleavage of dilute solutions of thymine dimer at 25†. It is suggested that these compounds might prevent the orientation of thymine molecules necessary for their photoactivation. These findings stress the potential role of the surrounding medium in photoreactions involving DNA both in vitro and in vivo.