Xanthine

Abstract: The reduction of cytochrome c by xanthine oxidase and the competitive inhibition of this process by carbonic anhydrase and by myoglobin have been studied by kinetic and by equilibrium binding methods. Carbonic anhydrases isolated from bovine and from human erythrocytes differed strikingly in their ability to inhibit competitively the reduction of cytochrome c. The Ks for cytochrome c was a function of the concentration of xanthine oxidase, as were Ki for carbonic anhydrase and Ki for myoglobin, whereas Ks for xanthine was invariant under the same conditions. Binding studies performed by a variety of methods indicated that carbonic anhydrase does not bind to xanthine oxidase. Carbonic anhydrase was found to be a potent inhibitor of the sulfite-oxygen chain reaction initiated either by the reduction of oxygen at an electrode or by xanthine oxidase plus xanthine. The data are consistent with the conclusion that xanthine oxidase, when catalyzing the aerobic oxidation of xanthine, generates an unstable reduced form of oxygen, presumably the superoxide anion, and that this radical is the agent which directly reduces cytochrome c and initiates the sulfite-oxygen chain reaction. Carbonic anhydrase and myoglobin appear to inhibit the reduction of cytochrome c and the initiation of sulfite oxidation by reducing the steady state concentration of the superoxide anion. It is proposed that they accomplish this end by catalyzing the following dismutation reaction: O2·− + O2·− + 2H+ → O2 + H2O2

Abstract: Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.

Abstract: Introduction 1 Principle of the method 1 Brief background of purine metabolism in ruminants 2 Limitation of the method 5 Sample collection 5 Determination of purine derivatives 6 Dilution of urine samples 6 List of published methods 8 Determination of allantoin by a colorimetric method 9 Determination of xanthine plus hypoxanthine by enzymatic method 12 Determination of uric acid by uricase method 15 Calculations 16 Daily excretion of purine derivatives 17 Calculation of microbial N supply 17 Presentation of results 18 Use of spot samples 19 Related Literature 19

Abstract: At pH 7.0, in air, 20% of the total electron flux through xanthine oxidase can be accounted for in terms of the univalent reduction of oxygen. The fraction of the total flux of electrons which traversed the univalent pathway to oxygen was increased by raising the pH and by raising the oxygen tension. It was further shown that at any given pH and oxygen tension, the amount of univalently reduced oxygen, which was detectable in terms of the reduction of cytochrome c, rose as the turnover rate of the enzyme was decreased by decreasing the concentration of xanthine. This effect of xanthine was more pronounced at pH 7.0 than at pH 10.0. Another reflection of this same phenomenon was a difference in Km for xanthine measured in terms of urate production as compared to Km for xanthine measured in terms of cytochrome c reduction. Here too the differences were diminished as the pH and the oxygen tension were raised. The quantitative aspects of these phenomena are presented as well as an explanation which is consistent with all of the observations and which was, in fact, predictive of several of them.

Abstract: Nitric oxide (NO.) is a physiological messenger formed by several cell types. Reaction with O2 forms oxides that nitrosate amines at pH values near 7. We now report experiments in which NO. was added to intact human cells and to aerobic solutions of DNA, RNA, guanine, or adenine. TK6 human lymphoblastoid cells were mutated 15- to 18-fold above background levels at both the HPRT and TK gene loci. Xanthine and hypoxanthine, from deamination of guanine and adenine, respectively, were formed in all cases. NO. induced dose-responsive DNA strand breakage. Yields of xanthine ranged from nearly equal to about 80-fold higher than those of hypoxanthine. Yields of xanthine and hypoxanthine from nucleic acids were higher than those from free guanine and adenine. This was most pronounced for xanthine; 0.3 nmol/mg was formed from free guanine vs. 550 nmol/mg from calf thymus RNA. Nitric oxide added to TK6 cells produced a 40- to 50-fold increase in hypoxanthine and xanthine in cellular DNA. We believe that these results, plus the expected deaminations of cytosine to uracil and 5-methylcytosine to thymine, account for the mutagenicity of nitric oxide toward bacteria and mammalian cells.