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.     

The effect of doxycycline on the intestinal alkaline phosphatase (EC 3.1.3.1) activity in rat. A quantitative study

The doxycycline effect on the alkaline phosphatase activity in the small intestinal glandular epithelium in rat was examined, using the interferometric technique. The antibiotic caused a statistically significant fall of the alkaline phosphatase activity and the enzyme was found to decrease with consecutive doses of the drug.     

Short communication: Purification and properties of acid phosphatase (EC 3.1.3.2) secreted by strain 74A of the mould Neurospora crassa

Both P_i-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mm ZnCl₂ and are non-specifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative co-operativity effect with a K_0.5 of 2.5 mm) whereas form IIb shows Michaelis kinetics, with a K_m of 0.5 mm. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium.     

Concerning the question of individual constancy and individual specificity of three serum enzyme activities (serum cholinesterase EC 3.1.1.8, ceruloplasmin EC 1.10.3.2, and alkaline phosphatase EC 3.1.3.1)

Summary Several assays of the enzymatic activities of serum cholinesterase, ceruloplasmin and alkaline phosphatase of 30 healthy students during a 9 months period revealed quite constant enzyme levels for each subject. Further, it was observed that most of the subjects maintained a specific enzyme activity value and that the individual range of scatter was characteristic for each one of them.According to our studies it seems probable that quantitative enzyme assays in adults can also be rated as biochemical parameters of individuality.Supported by the Deutsche Forschungsgemeinschaft.     

Kinetic properties of pyruvate kinase of Neurospora crassa at physiological pH.

The kinetic properties of pyruvate kinase (EC 2.7.1.40) extracted from the mycelia of Neurospora crassa were examined at physiological pH to determine the role of the enzyme in the regulation of glycolysis. The velocity curve with the substrates, phosphoenolpyruvate and adenosine diphosphate, are hyperbolic. The effect of magnesium, potassium, or calcium on the enzyme is influenced by the pH but not to the extent that would change their role as cofactor or inhibitor. Adenosine triphosphate and citrate remain strong inhibitors even with changes in pH. Fructose-1,6-diphosphate and glucose-6-phosphate are the dual positive effectors at physiological pH. Valine is the only amino acid that inhibits the enzyme at a concentration range of valine found in the mycelial juice. Thus, the properties of the enzyme at physiological pH are significantly different from those observed at neutral pH of the usual assay conditions, but its role as a key regulator of glycolysis is unchanged.     

Effect of cadmium on the activity of alkaline phosphatase (3.1.3.1) of Venus gallina]

The marine mollusc Venus gallina was exposed to 32 days sublethal concentrations of Cadmium (0.1 microliter/ml). The activity of alkaline phosphatase was assayed every four days. In the controls the activity was also tested in different tissues and in the soft tissue for the pH dependence. Moreover the influence of direct addition of 10(-4) M Cd on enzyme preparation in vitro was assayed. From the results there are no consistent relationships between the direct in vitro effects of the metal on the enzyme, that is inhibitory, and the effect of exposing the whole animal to the same metal, in this case no inhibition was observed.     

Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase.

Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.     

Isolation and properties of tripolyphosphatase from Neurospora crassa]

Homogenous preparation of tripolyphosphatase from Neurospora crassa is obtained. The enzyme is found to consist of two equal subunits with molecular weight of 40 000 and to have pH optimum 7.0 and temperature optimum 50 degrees C. Bivalent metal ions are required for its catalytical activity, the hest activators being Co2+, Mg2+ and Mn2+. Strict specificity of the enzyme to tripolyphosphate is demonstrated, Km being 5.9-10(-4) M. The enzyme hydrolyses tripolyphosphate to equimolar mixture of ortho- and pyrophosphate. The enzyme activity depends on orthophosphate and pyrophosphate concentrations in the incubation medium.