Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Altered glucose 1,6-bisphosphate and fructose 2,6-biphosphate levels in low-frequency stimulated rabbit fast-twitch muscle

Glucose 1,6-bisphosphate (Glc-1,6-P2) and fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations display pronounced increases in rabbit fast-twitch muscle during chronic low-frequency stimulation. These increases are first seen after stimulation periods exceeding 3 h and reach maxima after 12-24 h of stimulation (approximately 3-fold for Glc-1,6-P2 and 5-fold for Fru-2,6-P2). Both metabolites regress to normal values after stimulation periods longer than 4 days. The fact that their increases coincide with the replenishment of glycogen after its initial depletion, could point to a role of Glc-1,6-P2 and Fru-2,6-P2 in glycogen metabolism.     

Regulation of sugar transport in Neurospora crassa

Sugar uptake systems in Neurospora crassa are catabolically repressed by glucose. Synthesis of a low K(m) glucose uptake system (system II) in Neurospora is derepressed during starvation for an externally supplied source of carbon and energy. Fasting also results in the derepression of uptake systems for fructose, galactose, and lactose. In contrast to the repression observed when cells were grown on glucose, sucrose, or fructose, system II was not repressed by growth on tryptone and casein hydrolysate. System II was inactivated in the presence of 0.1 m glucose and glucose plus cycloheximide but not by cycloheximide alone. Inactivation followed first-order kinetics with a half-time of 40 min. The addition of glycerol to the uptake medium had no significant effect on the kinetics of 3-0-methyl glucose uptake, suggesting that the system was not feedback inhibitable by catabolites of glycerol metabolism.     

Regulation of hypoxanthine transport in Neurospora crassa.

Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.     

Regulation of a Sulfur-Controlled Protease in Neurospora crassa

Wild-type Neurospora crassa produces and secretes extracellular protease(s) when grown on a medium containing a protein as its principle sulfur source. Readily available sulfur sources, such as sulfate or methionine, repress the synthesis of the proteolytic activity. Preliminary characterization of the proteolytic enzyme shows it to have a molecular weight of about 31,000, a pH optimum of 6 to 9 with casein as substrate, and esterolytic activity against acetyl-tyrosine ethyl ester with a pH optimum of 8.5. The enzyme activity is completely inhibited by diisopropylfluorophosphate, partially inhibited by ethylenediaminetetraacetate, but unaffected by iodoacetate. The proteolytic activity is temperature labile and is reduced by 75% within 15 min at 60 C. Synthesis of the protease activity is induced by proteins, and to a lesser extent by large-molecular-weight polyamino acids, but not at all by small peptides or amino acid mixtures. During conidial out-growth, the protease(s) first appears at about 8 h and continues to increase while the cells are in an active growth phase. When a low concentration of sulfate is present, the protease(s) is not produced until about 18 h, suggesting that the sulfate must first be used by the cells before the protease is either synthesized or released.     

Regulation of thymidine metabolism in Neurospora crassa.

The utilization of thymidine by Neurospora crassa is initiated by the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and the consequent formation of thymine and ribose. Thymine must then be oxidatively demethylated by the thymine 7-hydroxylase and uracil-5-carboxylic acid decarboxylase reactions. This article shows that the 2'-hydroxylase reaction can be regulated differently than the oxidative demethylation process and suggests that the 2'-hydroxylase has, in addition to the role of salvaging the pyrimidine ring, the role of providing ribose not only for the utilization of the demethylated pyrimidine but also for other metabolic processes. One way that this difference in regulation was observed was with the uc-1 mutation developed by Williams and Mitchell. The present communication shows that this mutation increases the activities of the 7-hydroxylase and the decarboxylase but has no comparable effect on the 2'-hydroxylase. Qualitatively similar effects on these enzymes were bought about by growth of wild-type Neurospora in media lacking ammonium ion, such as the Westergaard-Mitchell medium. The 2'-hydroxylase and 7-hydroxylase are also differently affected by the carbon dioxide content of the atmosphere above the growing culture and the growth temperature. Studies with inhibitors indicated that the carbon dioxide effect is dependent on protein synthesis.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.     

Regulation of fructose 2,6-bisphosphate levels in cold-acclimated brown adipose tissue of rat

The effects of cold exposure and T4 administration on fructose 2,6-bisphosphate levels, phosphofructokinase-2 and pyruvate kinase activities were examined in rat brown adipose tissue. Cold adaptation (14 days) gave rise to a 2-fold increase in the amount of fructose 2,6-bisphosphate and phosphofructokinase-2 activity, and increased the pyruvate kinase activity 4-fold. If, in addition, the cold-acclimated rats were treated with T4, these parameters were again significantly enhanced. The effect on phosphofructokinase-2 was on the Vmax, without modification of the Km (for both fructose 6-phosphate and ATP) of the enzyme. In the hypothyroid state, however, the activity of pyruvate kinase remains unchanged. These data support previous observations on stimulation of glycolytic flux during cold adaptation in brown adipose tissue, and a permissive role of thyroid hormones in the process.     

Regulation of rat liver fructose 2,6-bisphosphatase

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.     

Regulation and function of glutamate synthase in Neurospora crassa

In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a$\text{m}$ monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am-1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.     

Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.