Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Microbial activity, arbuscular mycorrhizal fungi and inoculation of woody plants in lead contaminated soil

The goals of this study were to evaluate the microbial activity, arbuscular mycorrhizal fungi and inoculation of woody plants (Caesalpinia ferrea, Mimosa tenuiflora and Erythrina velutina) in lead contaminated soil from the semi-arid region of northeastern of Brazil (Belo Jardim, Pernambuco). Dilutions were prepared by adding lead contaminated soil (270 mg Kg-1) to uncontaminated soil (37 mg Pb Kg soil-1) in the proportions of 7.5 percent, 15 percent, and 30 percent (v:v). The increase of lead contamination in the soil negatively influenced the amount of carbon in the microbial biomass of the samples from both the dry and rainy seasons and the metabolic quotient only differed between the collection seasons in the 30 percent contaminated soil. The average value of the acid phosphatase activity in the dry season was 2.3 times higher than observed during the rainy season. There was no significant difference in the number of glomerospores observed between soils and periods studied. The most probable number of infective propagules was reduced for both seasons due to the excess lead in soil. The mycorrhizal colonization rate was reduced for the three plant species assayed. The inoculation with arbuscular mycorrhizal fungi benefited the growth of Erythrina velutina in lead contaminated soil.

Purification and characterization of a low-molecular-weight bovine kidney acid phosphatase

A relative low molecular mass bovine kidney acid phosphatase was purified 1,640-fold to homogeneity, with 7 percent recovery. The purified enzyme (specific activity 100 mumol min-1 mg-1) was electrophoretically homogeneous with a relative molecular massa of 17.8 kDa, as determined by SDS-polyacrylamide gel electrophoresis. A broad pH optimum of 4.0-5.5 and a maximal enzyme activity at 60 degrees Celsius were determined for the p-nitrophenyl phosphate hydrolysis. Apparent Km values of 0.14 mM, 0.4 mM, 0.3 mM and 7.9 mM were obtained, at 37 degrees Celsius and pH 5.0, for the best substrates p-nitrophenyl phosphate, beta-naphtyl-phosphate, flavin mononucleotide and tyrosine-phosphate, respectively. The enzyme activity was enhanced by guanosine but inhibited by ZnCl2 and CuSO4, p-cloromercuribenzoate and ammonium molybdate. Vanadate (Ki 0.47 muM), pyridoxal 5'-phosphate (Ki 2.2 muM), inorganic phosphate (Ki 0.77 mM) are competitive inhibitors. Both glycerol and methanol increased significantly the acide phosphatase activity, acting as good phosphate acceptors in the transphosphorylation reaction.

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Does gene palB regulate the transcription or the post-translational modification of Pi-repressible phosphatases of Aspergillus nidulans?

When grown on low-Pi medium, the chaA1 pabaA1 palB7 mutant of Aspergillus nidulans excretes an acid phosphatase with steady-state kinetic properties, temperature sensitivity and electrophoretic mobility different from those of the enzyme excreted by the pabaA1 strain. The enzyme excreted by the pabaA1 strain at pH 6.5 showed PNP-P activity with negative cooperativity (K0.5 = 0.87 + or - 0.06 mM, n = 0.68 + or - 0.03) whereas the enzyme excreted by the chaA1 pabaA1 palB7 mutant showed Michaelian kinetics (Km = 0.46 + or - 0.03 mM, n = 1.00 + or - 0.02). The apparent half-lives at 60§C, pH 5.5, of acid phosphatase excreted by the pabaA1 and chaA1 pabaA1 palB7 strains were 58.6 + or - 4.9 min and 21.5 + or - 1.8 min, respectively. Furthermore, the electrophoretic mobility of acid phosphatases excreted by the palA1, palB7, palC4, palE11 and palF15 mutants of A. nidulans was altered and differed from the electrophoretic mobility of the enzyme excreted by the wild-type strain. Also, the palB7 mutation altered the electrophoretic pattern of acid phosphatases synthesized on high-Pi medium. These results are compatible with the post-translational modifications in the Pi-repressible phosphatases rather than with the action of gene palB in controlling the transcription of structural genes of these enzymes

Partial purification of acid phosphomonoesterase in axenically grown Entamoeba histolytica NIH-200.

Entamoeba histolytica possesses significant acid phosphatase activity as compared to alkaline phosphatase activity. The acid phosphatase activity in the amoebic cells eluted at higher saline concentration as three distinct peaks at 200, 300 and 400 mM sodium chloride.