Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Properties of a D-glutamic acid-requiring mutant of Escherichia coli

Some properties of a d-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of d-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-l-alanine (UDP-MurNac-l-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing l-[(14)C]alanine. After incubation of the parental strain in a cell wall medium containing l-[(14)C]glutamic acid, the acid-precipitable radioactivity was lysozyme degradable to a large extent. Radioactive UDP-MurNac-pentapeptide was isolated from the l-[(14)C]glutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo-isomeric d-configuration. Compared to the parent the mutant incorporated less l-[(14)C]glutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide precursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNac-l-Ala:d-glutamic acid ligase (ADP; EC 6.3.2.9) (d-glutamic acid adding enzyme) is not affected by the mutation. Possible pathways for d-glutamic acid biosynthesis in E. coli B are discussed.     

Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage

Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential sigma(54)-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the sigma(54)-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced (14)CO(2) from minimal medium containing [(14)C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated (14)CO(2) production from [(14)C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells was monitored using 8-14C-adenosine and 8-14C-adenine added to the incubation medium containing adenosine, pyruvate and inorganic phosphate (APP medium). Using 8-14C-adenosine it was shown that 21.7% of the isotope contained in the incubation medium penetrated red blood cells. Of that quantity about 50% becomes incorporated into nucleotides. Of the isotope 5.3% was found in hypoxanthine nucleotides (1.3% in ITP and 4.0% in IMP). During incubation of red blood cells in APP medium fortified with the 8-14C-adenine about 95% of isotope penetrated into cells and 60% of that quantity became incorporated into nucleotides. In hypoxanthine nucleotides only trace amounts of isotope were found (0.12% in IMP and 0.13% in ITP).     

Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells.

Using the S49 T-cell lymphoma system for the study of immunodeficiency diseases, we characterized several variants in purine salvage and transport pathways and studied their responses to the cytotoxic action of adenosine (5-20 micron) in the presence of adenosine deaminase (ADA) inhibitors. Both an adenosine transport deficient mutant and a mutant lacking adenosine (ado) kinase activity are resistant to the cytotoxic effects of adenosine up to 15 micron. Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase are sensitive to the killing action of adenosine. We monitored the intracellular concentrations of purine and pyrimidine nucleotides, orotate, and PPriboseP in mutant and wild-type cells following the addition of adenosine and an ADA inhibitor. We conclude that at low concentrations, adenosine must be phosphorylated to deplete the cell of pyrimidine nucleotides and PPriboseP and to promote the accumulation of orotate. These alterations account for one mechanism of adenosine toxicity.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Utilization by Saccharomyces cerevisiae of 5'-methylthioadenosine as a source of both purine and methionine.

Cells of the yeast Saccharomyces cerevisiae are normally impermeable to the purine nucleosides adenosine and 5'-deoxy-5'-methylthioadenosine (MTA), a product of polyamine biosynthesis. cordycepin-sensitive, adenosine-utilizing strains of S. cerevisiae were able to use MTA to fulfill an auxotrophic requirement for purine. Cordycepin-sensitive strains carrying a met5 mutation were also able to use MTA as a source of methionine. These MTA-utilizing strains of S. cerevisiae should be useful for metabolic studies of the fate of MTA.     

Involvement of Polyamine Binding Protein D (PotD) of Synechocystis sp. PCC 6803 in Spermidine Uptake and Excretion

The in vivo function of polyamine binding protein D (PotD) in Synechocystis sp. PCC 6803 for the transport of spermidine was investigated using Synechocystis mutant disrupted in potD gene. The growth rate of potD mutant was similar to that of wild-type when grown in BG11 medium. However, the mutant exhibited severely reduced growth compared to the wild-type when BG11 medium was supplemented with 0.5 mM spermidine. The mutant accumulated a higher spermidine level than the wild-type when grown in the medium with or without spermidine. Transport experiments revealed that the mutant had a reduction in both the uptake and the excretion of spermidine. Moreover, [¹⁴C]spermidine-loaded wild-type and mutant cells showed a decrease of [¹⁴C]spermidine excretion when the assay medium contained exogenous spermidine. These data suggest that PotD is involved in both the uptake and the excretion of spermidine in Synechocystis cells.     

Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Adenosine phosphorylase activity in a mutant Hep-2 cell line contaminated withmycoplasma hyorhinis

Summary Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor coformycin. Isopycnic separation of [³H]thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [¹⁴C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified asMycoplasma hyorhinis, a porcine mycoplasma. Following γ-irradiation (3000 rads) to block cellular mitosis, the mycoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma. Presented at the 25th Annual Meeting of the Tissue Culture Association, Philadelphia, Pa., June 1976. This work was supported by Public Health Service Grants DE 02731 from the National Institute of Dental Research and CA 16219 from the National Cancer Institute.     

Adenosine phosphorylase activity in a mutant HEp-2 cell line contaminated with Mycoplasm hyorhinis.

Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor conformycin. Isopycnic separation of [3H] thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [14C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified as Mycoplasma hyorhinis, a porcine mycoplasma. Following gamma-irradiation (3000 rads) to block cellular mitosis, the mucoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma.     

Purine base transport in Neurospora crassa.

Observations presented in this paper point to the presence of dual transport mechanisms for the base adenine in Neurospora crassa. Competition for transport, as well as growth inhibition studies using an ad-1 auxotroph, show that the purine bases adenine, guanine, and hypoxanthine share at least one transport mechanism which is insensitive to adenosine, cytosine, and a variety of other purine base analogues. On the other hand, uptake of adenine by an ad-8 mutant strain unable to transport [8-14C]hypoxanthine at any concentration was not inhibited by guanine or hypoxanthine. This observation demonstrates the existence of an adenine-specific transport system which was also found to be insensitive to inhibition by other purine base analogues, adenosine or cytosine. Recombination analysis of ad-8 by wild-type crosses showed that the inability to transport [8-14C]hypoxanthine was a consequence of the ad-8 lesion or a closely linked mutation. Saturation plots of each system gave intermediary plateaus and nonlinear reciprocal plots which, based on comparison with pure enzyme kinetic analysis, suggest that either each system consists of two or more uptake systems, at least one of which exhibits cooperativity, or that each system is a single uptake mechanism which possesses more than two binding sites where the relative affinity for the purine base first decreases and then increases as the sites are filled.     

Growth Properties of Rhodospirillum rubrum Mutants and Fermentation of Pyruvate in Anaerobic, Dark Conditions

Mutant C and G1 were obtained earlier from Rhodospirillum rubrum S(1) during growth in the dark under strict anaerobic conditions in medium containing sodium pyruvate. Mutant C and mutant G1 grew in the dark with generation times of 5.8 h and 4.6 h, respectively. Mutant C cells grew equally well when switched between anaerobic (dark or light) or aerobic, dark conditions. Mutant G1 cells grew only in the dark (anaerobic or aerobic conditions), but a fraction of cells in anaerobic, dark cultures grew when placed in light. This number increased about 3,000-fold when G1 cells were incubated aerobically in the dark. During anaerobic, dark growth, C and G1 organisms incorporated similar amounts of [2-(14)C]sodium pyruvate. About 34% of the incorporated radioactivity was found in lipid fractions from C cells that developed chromatophores during dark growth. Similar results were obtained using G1 cells, which formed only trace amounts of photosynthetic structures. Both mutants fermented sodium pyruvate and produced acetate, formate, carbon dioxide, and hydrogen gas. Molar growth yield coefficients indicated that the cells obtained about 1 mol of adenosine triphosphate per mol of sodium pyruvate fermented. Results suggested that pyruvate fermentation during dark growth occurred via a pyruvate formate-lyase or the pyruvate ferredoxin-oxidoreductase pathway, or both.     

Adenosine kinase-deficient mutant of Neurospora crassa.

Tubercidin-resistant mutant strains of Neurospora crassa were isolated, and at least one appeared to be deficient in adenosine kinase. No significant differences in [8-14C]adenosine labeling of purine nucleotides or nucleosides were found between the wild type and the adenosine kinase-deficient strains.