Genetic and metabolic regulation of purine base transport in Neurospora crassa.

Summary Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. The early purine catabolic enzymes in this organism are regulated by induction and by ammonium repression. Studies were undertaken to investigate purine base transport and its regulation in Neurospora. The results of competition experiments with uric acid and xanthine transport strongly suggest that uric acid and xanthine share a common transport system. It was also shown that the common transport system for uric acid and xanthine is distinct from a second transport system shared by hypoxanthine, adenine and guanine, and apparently also distinct from the transport system(s) for adenosine, cytosine and uracil. Regulation of the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system was studied. The results reveal that the uric acid-xanthine transport system is regulated by ammonium repression, but does not require uric acid induction. Neither ammonium repression nor uric acid induction controls the hypoxanthine-adenine-guanine transport system. A gene, designated amr, which is believed to be a positive regulatory gene for nitrogen metabolism of Neurospora crassa, was found to dramatically affect both the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system. A model for the action of the amr locus as a positive regulatory gene and for the interaction between the amr gene product and its recognition sites will be discussed.     

Role of protein degradation in the regulation of cellular protein content and amino acid pools

A decrease in the rate of protein degradation contributes to the accumulation of cellular protein during growth or storage of dietary amino acids. Examples of this process in the liver of live mice are reviewed. One aspect of this regulation is the direct effect of amino acids on the rate of protein breakdown. At least in the case of histidine starvation in Chinese hamster ovary cells, the regulatory mechanism recognizes the level of aminoacylation of tRNA.     

Major determinants of purine excretion from human lymphoblasts

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

Bacterial protein meal in diets for pigs and minks: Comparative studies on protein turnover rate and urinary excretion of purine base derivatives

Abstract

The effect of increasing the dietary content of bacterial protein meal (BPM) on protein turnover rate, and on nucleic acid and creatinine metabolism in growing minks and pigs was investigated in two experiments. In each experiment, 16 animals were allocated to four experimental diets. The diets containing no BPM served as controls, i.e. for minks diet M1, for pigs P1; the experimental diets contained increasing levels of BPM to replace fish meal (minks) or soybean meal (pigs), so that up to 17% (P2), 20% (M2), 35% (P3), 40% (M3), 52% (P4), and 60% (M4) of digestible N was BPM derived. Protein turnover rate was measured by means of the end-product method using [¹⁵N]glycine as tracer and urinary nitrogen as end-product. In minks, protein flux, synthesis, and breakdown increased significantly with increasing dietary BPM. In pigs, diet had no observed effect on protein turnover rate. The intake of nucleic acid nitrogen (NAN) increased from 0.15 g/kg W^0.75 on M1 to 0.26 g/kg W^0.75 on M3 and M4 in the mink experiment, and from 0.08 g/kg W^0.75 on P1 to 0.33 g/kg W^0.75 on P4 in the pig experiment. Increased NAN intake led, in both experiments, to increased allantoin excretion. Analysis of species effects showed that minks excreted 1.72 mmol/kg W^0.75 of allantoin, significantly more than the 0.95 mmol/kg W^0.75 excreted by pigs. In minks, approximately 96% of the excreted purine base derivatives consisted of allantoin, whereas in pigs approximately 93% did. Thus, increasing the dietary content of BPM increased protein turnover rate in minks but not in pigs, and allantoin excretion increased with increasing dietary BPM although it seemed that mink decomposed purine bases to their end-product more completely than pigs did. Collectively these data show that BPM is a suitable protein source for pigs and mink, and recorded differences between species were to a large extent due to differences in protein retention capacity and muscle mass.     

Bacterial protein meal in diets for pigs and minks: comparative studies on protein turnover rate and urinary excretion of purine base derivatives

The effect of increasing the dietary content of bacterial protein meal (BPM) on protein turnover rate, and on nucleic acid and creatinine metabolism in growing minks and pigs was investigated in two experiments. In each experiment, 16 animals were allocated to four experimental diets. The diets containing no BPM served as controls, i.e. for minks diet M1, for pigs P1; the experimental diets contained increasing levels of BPM to replace fish meal (minks) or soybean meal (pigs), so that up to 17% (P2), 20% (M2), 35% (P3), 40% (M3), 52% (P4), and 60% (M4) of digestible N was BPM derived. Protein turnover rate was measured by means of the end-product method using [15N]glycine as tracer and urinary nitrogen as end-product. In minks, protein flux, synthesis, and breakdown increased significantly with increasing dietary BPM. In pigs, diet had no observed effect on protein turnover rate. The intake of nucleic acid nitrogen (NAN) increased from 0.15 g/kg W0.75 on M1 to 0.26 g/kg W0.75 on M3 and M4 in the mink experiment, and from 0.08 g/kg W0.75 on P1 to 0.33 g/kg W0.75 on P4 in the pig experiment. Increased NAN intake led, in both experiments, to increased allantoin excretion. Analysis of species effects showed that minks excreted 1.72 mmol/ kg W0.75 of allantoin, significantly more than the 0.95 mmol/kg W0.75 excreted by pigs. In minks, approximately 96% of the excreted purine base derivatives consisted of allantoin, whereas in pigs approximately 93% did. Thus, increasing the dietary content of BPM increased protein turnover rate in minks but not in pigs, and allantoin excretion increased with increasing dietary BPM although it seemed that mink decomposed purine bases to their end-product more completely than pigs did. Collectively these data show that BPM is a suitable protein source for pigs and mink, and recorded differences between species were to a large extent due to differences in protein retention capacity and muscle mass.     

Role of orthophosphate concentration in the regulation of ribose phosphate synthesis and purine metabolism in Ehrlich ascites tumor cells

Concentrations of intracellular orthophosphate were determined in Ehrlich ascites tumor cells incubated with glucose, inosine, or uridine in media of different orthophosphate concentration. The effects of orthophosphate concentration on the accumulation of lactate and of phosphoribosyl pyrophosphate and on concentrations of ribose 1-phosphate and ribose 5-phosphate in tumor cells incubated with glucose were also determined. Both the phosphorolysis of inosine and the rate of catabolism of ATP in cells incubated with 2-deoxyglucose were also influenced by the orthophosphate concentration of the medium.     

Synapsin regulation of vesicle organization and functional pools

Synaptic vesicles are organized in clusters, and synapsin maintains vesicle organization and abundance in nerve terminals. At the functional level, vesicles can be subdivided into three pools: the releasable pool, the recycling pool, and the reserve pool, and synapsin mediates transitions between these pools. Synapsin directs vesicles into the reserve pool, and synapsin II isoform has a primary role in this function. In addition, synapsin actively delivers vesicles to active zones. Finally, synapsin I isoform mediates coupling release events to action potentials at the latest stages of exocytosis. Thus, synapsin is involved in multiple stages of the vesicle cycle, including vesicle clustering, maintaining the reserve pool, vesicle delivery to active zones, and synchronizing release events. These processes are regulated via a dynamic synapsin phosphorylation/dephosphorylation cycle which involves multiple phosphorylation sites and several pathways. Different synapsin isoforms have unique and non-redundant roles in the multifaceted synapsin function.     

A comparison of purine metabolism and nucleotide pools in normal and hypoxanthine-guanine phosphoribosyltransferase-deficient neuroblastoma cells

Purine nucleotide synthesis and interconversion were examined over a range of purine base and nucleoside concentrations in intact N4 and N4TG (hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient) neuroblastoma cells. Adenosine was a better nucleotide precursor than adenine, hypoxanthine or guanine at concentrations greater than 100 μM. With hypoxanthine or guanine, N4TG cells had less than 2% the rate of nucleotide synthesis of N4 cells. At substrate concentrations greater than 100 μM the rates for deamination of adenosine and phosphorolysis of guanosine exceeded those for any reaction of nucleotide synthesis. Labelled inosine and guanosine accumulated from hypoxanthine and guanine, respectively, in HGPRT-deficient cells and the nucleosides accumulated to a greater extent in N4 cells indicating dephosphorylation of newly synthesized IMP and GMP to be quantitatively significant. A deficiency of xanthine oxidase, guanine deaminase and guanosine kinase activities was found in neuroblastoma cells. Hypoxanthine was a source for both adenine and guanine nucleotides, whereas adenine or guanine were principally sources for adenine (>85%) or guanine (>90%) nucleotides, respectively. The rate of [¹⁴C]formate incorporation into ATP, GTP and nucleic acid purines was essentially equivalent for both N4 and N4TG cells. Purine nucleotide pools were also comparable in both cell lines, but the concentration of UDP-sugars was 1.5 times greater in N4TG than N4 cells.     

Urinary excretion of purines,purine nucleosides,and pseudouridine in immunodeficient children

Studies have been carried out using ion-exchange analysis for determination of urinary purines, pyrimidines, and nucleosides in children with immunodeficiency disorders. Using cation and anion-exchange techniques, the following compounds of urine have been quantitatively determined: deoxyadenosine, adenosine, adenine, pseudouridine, 7-methylguanine, N²,N²-dimethylguanosine, hypoxanthine, and xanthine. Excretion levels of these compounds did not differ significantly from normal values in six children with various immunodeficiency diseases, excluding severe combined immunodeficiency. However, of the seven children studied with severe combined immunodeficiency disease (normal adenosine deaminase), four showed increased excretion levels for one or more of the compounds studied. A germ-free child with severe combined immunodeficiency had lower excretion levels than the mean normal value for most of these same compounds. The possibility is considered that the increased excretion levels noted may be a consequence of repeated episodes of infection in most children with severe combined immunodeficiency.     

Role of prostaglandin E2 in regulation of urine excretion in saluresis, water and osmotic diuresis in rat

In the saluresis, water and osmotic diuresis were indicating an increase of prostaglandin E2 excretion and a correlation between this index and diuresis. Unselective blockade of cyclooxygenase by diclofenac-natrium leads to a decrease of diuresis in the observed types of urine-production in rats. Inhibition of inducible cyclooxygenase by celebrex didn't change the value of diuresis after water load or administration of osmotic agent, but decreased the diuretic effect of furosemide.