Donald W. Visser

Studies on nitrate reduction in higher plants. I.

Abstract 1. 1. The distribution of N 15 followed the same pattern in all organs of intact plants, whether the nitrogen source was N 15 -labeled ammonium or N 15 -labeled nitrate; the ammonia fraction contained the highest atom per cent excess of N 15 in all cases. 2. 2. Plants which received ammonium contained the highest concentration of N 15 in the roots, whereas those which received nitrate contained the highest concentration in the leaves. 3. 3. Tomato leaf disks are able to assimilate nitrate in both the light and dark, but the rate of assimilation in the light is about 50% greater than in the dark. 4. 4. Carbohydrate depletion in leaf tissue results in a decreased nitrate reduction in the dark, whereas it is without effect in the light. 5. 5. Addition of oxidizable substrates to carbohydrate-depleted leaf tissue increased the dark reduction of nitrate, although not to the level of the fresh tissue. 6. 6. Iodoacetate markedly inhibits the reduction of nitrate in leaf disks incubated in the dark, but has no effect on the process in the light. 7. 7. Ammonia isolated from leaf disks incubated 30 min. in the light contained an N 15 enrichment almost two-thirds that of the original nitrate. The ammonia from the dark reaction was not so highly labeled, containing only about one-seventh the enrichment of N 15 of the medium. 8. 8. Evidence for two mechanisms of nitrate assimilation are obtained: one dependent upon respiration as an energy source, and the other involving a photochemical reduction.

Studies on 5-aminodeoxyuridine

Abstract 1. 1. Incorporation of isotope from [3- 14 C]serine into DNA thymine is inhibited in Escherichia coli K12 grown in presence of 5-aminodeoxyuridine and an exogenous source of thymidine to restore normal growth. Incorporation of isotope from the same source into DNA adenine was not effected. This was interpreted as evidence that 5-aminodeoxyuridine inhibits thymidylate synthetase. 2. 2. 5-aminodeoxyuridine potentiates the inhibitory effect of 5-fluorodeoxyuridine on the growth of E. coli K12, indicating that 5-aminodeoxyuridine inhibits the metabolism of thymine compounds subsequent to the formation of thymidine-monophosphate. Thus, 5-aminodeoxyuridine inhibits sequential reactions of thymidine metabolism namely, thymidylate formation and utilization of thymidylate for DNA or cofactor synthesis. 3. 3. A modification of the method of synthesis of 5-aminodeoxyuridine is described which increases the previously reported yield approx. seven-fold.

Transport studies of showdomycin, nucleosides and sugars in Escherichia coli B and in showdomycin-resistant mutants

Competition between the nucleoside antibiotic, showdomycin, and certain naturally occurring nucleosides for a common transport system can explain the protective action of the nucleosides against inhibition by showdomycin of growth, amino acid transport and sugar transport in Escherichia coli B. Uridine interferes with the transport of showdomycin into the cell, thereby preventing the inhibitory effects of the antibiotic. With the exception of adenosine, nucleosides which protect against inhibition by showdomycin are mutually competitive for their transport. Guanosine, which does not protect against the inhibitory effect of showdomycin on glucose transport, does not compete for the transport of the protective nucleosides, uridine, cytidine or adenosine. Adenosine inhibits transport of uridine, cytidine and guanosine, but appears to have separate transport requirements. A mutant of E. coli B resistant to 80 μM showdomycin exhibits normal growth and glucose transport properties. The transport of uridine, cytidine and showdomycin is markedly reduced in the mutant, whereas guanosine and adenosine transport is similar to that in wild-type cells. Exogenous showdomycin, which inhibits transport of glucose, guanosine and protective nucleosides in E. coli B, has no effect on these processes in mutant cells. A defect in the capacity of the mutant to transport showdomycin explains the resistance of the mutant to the inhibitory effects of showdomycin. The inhibitory effects of N-ethylmaleimide, in contrast to showdomycin, are similar in mutant and E. coli B cells.

Studies on 5-aminouridine

Abstract Inhibitory effects of 5-aminouridine and 5-aminouridylic acid were studied in Ehrlich ascites cells and cell-free preparations. The metabolism of synthetically prepared [2- 14 C]5-aminouridine was compared with that of [2- 14 C]uridine. The radioactive nucleoside analogue was metabolized to 5-amino-UMP in Ehrlich ascites cells and evidence for the presence of 5-amino-UDP, 5-amino-UTP and 5-amino-UDP-sugars in the acid-soluble fraction was obtained. Incorporation of analogue into RNA was proven by isolation of radioactive 5-aminouridine 2′(3′)-monophosphate from an alkaline hydrolysate of RNA isolated from the cells. The presence of 5-aminouridine 5′-phosphate inhibited formation of uridine nucleotides from [2- 14 C]orotic acid and caused accumulation of orotidylic acid in cell-free preparations from Ehrlich ascites cells. The data demonstrate that 5-aminouridine 5′-monophosphate is a potent inhibitor of orotidine-5′-phosphate decarboxylase (EC 4.1.1.23). Ehrlich ascites cells were incubated in the presence of equal amounts of either [2- 14 C]uridine or [2- 14 C]5-aminouridine to determine quantitative differences in the metabolism of the two compounds. Differences in the amounts of [2- 14 C]uridine and [2- 14 C]5-aminouridine in the respective acid-soluble nucleotides were no greater than 2-fold. The amount of [2- 14 C]uridine incorporated into nucleic acids, however, was 6.7 times greater than that of [2- 14 C]5-aminouridine.

Transport of uridine in Escherichia coli B and A showdomycin-resistant mutant

Abstract The mechanism of uridine transport in Escherichia coli B cells was studied using experimental approaches designed to limit possible ambiguities in interpretation of data obtained previously. For this purpose, the transport of [2-14C]uridine and [U-14C]uridine was determined in E. coli B and an E. coli B mutant which is resistant to the inhibitory effects of the nucleoside antibiotic, showdomycin. The majorty of the uridine transported as the intact nucleoside is cleaved to uracil and ribose l-phosphate. The uracil, in large part, is excreted, while ribose l-phosphate is retained. In addition, uridine is also rapidly cleaved to uracil and ribose l-phosphate in the periplasmic space. The uracil moiety may enter the cell, whereas ribose l-phosphate is not transported. The showdomycin-resistant mutant transports the intact nucleoside inefficiently, or not at all, but retains its ability to convert uridine to uracil in the periplasmic space.

Inhibition of Mouse Encephalomyelitis Virus, in vitro, by Certain Nucleoprotein Derivatives.∗

Summary Adenine, adenosine, cytidine, guanosine and thymine inhibit the propagation of Theilers GDVII strain of murine encephalomyelitis virus in tissue cultures of one-day mouse brain. Guanine, uridine, uracil, guanylic or cytidylic acids did not inhibit. Various substituted nucleosides including amino-, chloro-, diazo-, formamido-, hydroxy- and methyluridine were inhibitory. Uridine partially reversed this inhibition. 5-Chlorouracil, ribosylthymine and glucosylthymine did not inhibit; 2,6-diaminopurine did. 5-Chlorouridine was not effective against this viral infection in mice.

Inhibition of amino acid and sugar transport by showdomycin.

Abstract Showdomycin inhibits the uptake of sugars and amino acids in Escherichia coli B cells. The data show that inhibition of transport by the alkylating action of showdomycin is a primary explanation for its inhibitory effects on growth of E. coli . The inhibitory effects are reversed completely by preincubation with cysteine or common nucleosides excepting guanosine, deoxyguanosine and pseudouridine. Uridine produces a two- to three-fold increase of 2-deoxyglucose and α-methyl- d -glucoside transport into E. coli cells. Analysis of products accumulated from 2-deoxyglucose shows a greater increase of free sugar than the sugar phosphate.

Chemical Inhibitors of Theiler's Virus.

Summary Certain pyrimidine-related-com-pounds inhibit Theilers virus, in vitro. Inhibition by 5-hydroxyuridine is partially reversed by uridine-5′-phosphate. Of various other chemicals found to inhibit virus, in vitro, chlorpromazine and polymyxin did not protect mice from viral infection.

Uridine diphosphate glucose dehydrogenase of calf liver. Properties and inhibition characteristics with uridine diphosphate xylose analogues.

Abstract UDP-glucose dehydrogenase of calf liver dissociated in guanidine-HCl into six subunits. The number of reactive sulfhydrylgroups of native and guanidine-HCl-treated enzyme was found to be 20 ± 1 and 46 ± 1, respectively, per mole of native enzyme. A crude preparation of UDP-glucose pyrophosphorylase from yeast was used for the preparation of 5-hydroxyuridine diphosphate xylose and 5-aminouridine diphosphate xylose. 5,6-Dihydrouridine diphosphate xylose was prepared by catalytic reduction of UDP-xylose. The nature of the inhibition produced by UDP-xylose or its analogues was similar with respect to either UDP-glucose or NAD + , the two substrates for the enzyme. Hydrogenation of the 5,6-double bond or substitution of a hydroxyl group at the C-5 position of the pyrimidine portion of UDP-xylose decreased its inhibitory activity. Substitution of an amino group at the C-5 position, however, did not alter the activity of the allosteric inhibitor.

Uridine and uracil transport in Escherichia coli and transport-deficient mutants

Mutants of Escherichia coli K-12 which are defective in components of transport systems for uracil and uridine were isolated and utilized to characterize the transport mechanism of uracil and uridine. Mutant U-, isolated from a culture of the parent strain, is resistant to 5-fluorouracil and is deficient in the uracil transport system. Mutant UR-, isolated from a culture of the parent strain, is resistant to a low concentration of showdomycin and lacks the capacity to transport intact uridine. Mutant U-UR- isolated from a culture of mutant U-, is resistant to a low concentration of showdomycin and is defective in both uracil and intact uridine transport processes. Mutant UR-R- was isolated from a culture of mutant UR-, and is resistant to a high concentration of showdomycin. This mutant is defective for transport of intact uridine and addition lacks the transport system for the ribose moiety of uridine. Characteristics of uracil and uridine transport in parent and mutant cells demonstrate the existence of specific transport processes for uracil, intact uridine and the uracil and ribose moieties of uridine. Mutants U- and UR-, which are defective for uracil transport, lack uracil phosphoribosyltransferase activity and retain a small but significant capacity to transport uracil. The data support the conclusion that uracil is transported by two mechanisms, the major one of which requires uracil phosphoribosyltransferase activity, while the other process involves the transport of uracil as such. The characteristics of uridine transport in parent and mutant strains show that, in addition to transport as the intact nucleoside, uridine is rapidly cleaved to the uracil and ribose moieties. The latter is transported into the cell by a process which, in contrast to transport of intact uridine, does not require an energy source. The uracil moiety is released into the medium and is transported by the uracil transport system. Whole cells of the parent and mutant strains differ in their ability to cleave uridine even though cell-free extracts of all the strains have similar uridine phosphorylase activity. The data implicate a uridine cleavage enzyme in a group transport of the ribose moiety of uridine, a process which is nonfunctional in mutants which lack the capacity to transport the ribose moiety of uridine. A common transport component for this process and the transport of intact uridine is indicated by similarities in the inhibitory effects of heterologous nucleosides on these processes.