Robert S. Krooth

Studies on the Xanthine Oxidase Activity of Mammalian Cells

Xanthine oxidase in man is confined to but a few tissues and is absent from cultured cell strains. In rodents, however, the enzyme is more widely distributed among the tissues and can be demonstrated in most cell lines. Rodents possess the enzyme uricase and are therefore able to carry purine catabolism one step further than man. Preliminary results suggest that uricase is restricted to but a few rodent tissues and is absent from cultured rodent cells. Hence it may be that in each species only the final enzyme of purine catabolism is tissue restricted. In other experiments, mammalian cells were grown in the presence of compounds known to induce xanthine oxidase in a eukaryotic fungus (Aspergillus nidulans). These compounds did not induce the enzyme in mammalian cells.

Inhibition by barbituric acid and its derivatives of the enzymes in rat brain which participate in the synthesis of pyrimidine ribotides

Abstract Barbituric acid competitively inhibits dihydro-orotate dehydrogenase, uridine phosphorylase, and (directly or indirectly) orotate phosphoribosyltransferase. In addition, the ribotide of barbituric acid, 6-hydroxyuridine-5′-monophosphate, inhibits uridine monophosphate kinase and is a powerful competitive inhibitor of orotidylate decarboxylase—the final enzyme in the sequence for the de novo synthesis of pyrimidine ribotides. Barbituric acid itself causes marked competitive inhibition of decarboxylase activity provided the reaction mixture contains 5-phosphoribosyl-1-pyrophosphate (PRPP). Another barbituric acid derivative, phenobarbital, is a weak non-competitive inhibitor (in the presence of PRPP) of the decarboxylase. One convulsant barbiturate, DMBB [5-(1,3-dimethylbutyl)-5-ethyl-barbituric acid], and bemegride—a stimulant structurally resembling the barbiturates—were found to inhibit rat brain uridine phosphorylase activity, and this same enzyme was also inhibited by isobarbituric acid.

Determination of the intracellular concentration of 5-phosphoribosyl-1-pyrophosphate in cultured mammalian fibroblasts

Abstract The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of 14 CO 2 from [ carboxyl - 14 C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained 7.76 ± 1.14 nmol of PRPP 10 7 cells .

Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: A new bidirectional selective system for mammalian cells

We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible—in one experiment—to screen more than 1011 of these cells for growth under selective conditions. After exposure to a mutagen, descendants of our clone were placed in selective media containing 10−4 M 5-fluoroorotic acid and 10−4 M uridine. Cells capable of sustained growth in such media were eventually recovered. Clones of these cells, unlike clones of the parental population, required uridine for growth and contained only 0.39% as much orotate phosphoribosyltransferase, less than 0.5% as much 5-fluoroorotate phosphoribosyltransferase, and 0.013% as much orotidine-5′-monophosphate decarboxylase activity as the parental clones. The parental and variant clones had similar levels of activity for six other enzymes that participate in pyrimidine metabolism.

Effect of 6-azauracil, and of certain structurally similar compounds, on three pyridoxal-phosphate requiring enzymes involved in neurotransmitter metabolism☆

Abstract 6-Azauracil, which is reported to have an hypnotic effect in man, rat and mouse, is a noncompetitive inhibitor of gamma-aminobutyric acid (GABA)-transaminase activity in rodent and human brain extracts. The Ki for the rat brain activity is 5 × 10−4 M. GABA-transaminase is also non-competitively inhibited by a number of other compounds-both drugs and natural substances—which are reported to affect arousal and which, like 6-azauracil, include within their structure a pyrimidine or pyrimidine-like ring. None of these compounds, however, is as potent an inhibitor of the transaminase as 6-azauracil. Glutamic acid decarboxylase is inhibited only weakly (and non-competitively) by 6-azauracil and, to an even lesser extent, by the other substances tested. None of the compounds under study caused appreciable inhibition of DOPA decarboxylase except under conditions where the enzyme proved highly unstable, and even under these conditions the inhibition was weak.

Oxipurinol and orotic aciduria: Effect on the orotidine-5′-monophosphate decarboxylase activity of cultured human fibroblasts

Abstract Growth in certain pyrimidines or in oxipurinol, whose respective ribotides inhibit the final enzyme in the synthetic sequence leading to UMP, causes cultured cells to develop similar increases in activity for that enzyme. The increase is independent of the genotype of the cells for the known Mendelian mutations affecting the basal level of enzyme activity.

Studies of the orotidine 5′-monophosphate decarboxylase activity of crude extracts of human cells

The specific activity of orotidine 5′-monophosphate (OMP) decarboxylase in cultured human fibroblasts is an exponential function of the concentration of cell protein in the extract. Low concentrations of uridylic or cytidylic acid augment the catalytic activity of dilute solutions of cell extract but not of concentrated ones. In the presence of urea, specific activity becomes independent of protein concentration, and uridylic or cytidylic acid augments activity over all concentrations of cell extract. These results, as well as other observations, suggest that the decarboxylase may be composed of subunits which are in dynamic equilibrium with an aggregate. At least one of the subunits is likely to have catalytic activity for the reaction, though less activity than the aggregate. The effect of the mutant gene for orotic aciduria on OMP decarboxylase is easily demonstrated when cell extracts are assayed in either the presence or the absence of urea.

Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.

A deterministic mechanism for cellular and organismic ageing

Abstract The theory assumes that some genetic loci, encoding for enzymes, become serially repeated along the chromosome in the course of evolution. Ageing is supposed to involve a shift from a locus encoding for a functional protein to a locus which encodes for a similar but non-functional protein, or a protein with reduced or deleterious function. A shift from the appropriate aminoacyl tRNA synthetase to one which charges a species of tRNA with the wrong amino acid is particularly consistent with recent experimental results. Shifts in the expression of genetic loci encoding for similar proteins are known to occur in the early stages of mammalian development. A specific molecular mechanism for accomplishing very gradual shifts is reviewed, and a theory for the evolution of ageing, based on a supposed advantage ageing may confer on whole populations, is proposed.

A theory for the chemical mediation of the excitability of the brain, with special reference to natural and drug-induced sleep.

Abstract We review evidence which suggests to us that uracil, or a molecule structurally resembling uracil, is likely to be involved in the mediation of level of arousal. In addition we attempt to show that at least one theory, based on this idea, provides a molecular mechanism for certain features of natural sleep, and for the effect on arousal of several classes of drugs and of certain gene mutations. This theory, and related ones, can feasibly be tested further with existing methods.