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.     

The synthesis of alkaline phosphatase in Neurospora crassa

Mutations which affect the regulation of Neurospora repressible alkaline phosphatase do so by altering the rate of de novo alkaline phosphatase synthesis. In regulatory mutants the rate of alkaline phosphatase polypeptide synthesis can vary over a 1000-fold range. Following transfer to phosphate-free medium, the wild-type cell is capable of increasing the rate of synthesis of alkaline phosphatase molecules within 30-45 min.     

Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue.

Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.     

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.     

Nitrogen regulation of acid phosphatase in Neurospora crassa.

Neurospora crassa possesses a repressible acid phosphatase with phosphodiesterase activity which appears to permit it to utilize ribonucleic acid as a phosphorus and as a nitrogen source. This acid phosphatase, which is specified by the pho-3 locus, is derepressed approximately eightfold during nitrogen limitation and to an even greater extent during phosphorus limitation, but is unaffected by sulfur limitation. Derepression of the enzyme did not occur when adenosine 5'-monophosphate was the sole phosphorus or nitrogen source. Synthesis of the acid phosphatase is not under the control of the nit-2 locus, which regulates the expression of a large number of other nitrogen catabolic enzymes. The structural gene of the acid phosphatase appears to be a member of both the phosphorus and nitrogen regulatory circuits.     

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 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.