Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).     

Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (K(M) = 3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (Pi, Ki = 1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into Pi whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone. an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis (Ki = 60 microM) and co-mediates 18O-label exchange into Pi in a concentration-dependent manner (K(M) = 64 microM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.     

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.     

cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli

A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced. The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D. discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM). The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid).     

Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum

An acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme in gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 3.4 x 10(5) M-1 cm-1. The isoelectric point of the enzyme, as judged by chromatofocusing, was about 4.0. The purified enzyme is highly stable at 0 degree C. Thermal inactivation studies have indicated that the enzyme is unstable at 70 degrees C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63 degrees C. The Km of the enzyme for p-nitrophenylphosphate is about 270 microM with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-oligosaccharides. A partial N-terminal amino acid sequence up to the twenty-third residue was obtained. The enzyme was inhibited competitively by inorganic orthophosphate (Ki = 185 microM) and non-competitively by phosphomycin (Ki = 600 microM).     

Purification and characterization of vitamin B6-phosphate phosphatase from human erythrocytes.

Human erythrocytes rapidly convert vitamin B6 to pyridoxal-P and contain soluble phosphatase activity which dephosphorylates pyridoxal-P at a pH optimum of 6-6.5. This phosphatase was purified 51,000-fold with a yield of 39% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose, Sephacryl S-200, hydroxylapatite, and reactive yellow 86-agarose. Sephacryl S-200 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a dimer with a molecular mass of approximately 64 kDa. The phosphatase required Mg2+ for activity. It specifically catalyzed the removal of phosphate from pyridoxal-P, pyridoxine-P, pyridoxamine-P, 4-pyridoxic acid-P, and 4-deoxypyridoxine-P at pH 7.4. Nucleotide phosphates, phosphoamino acids, and other phosphorylated compounds were not hydrolyzed significantly nor were they effective inhibitors of the enzyme. The phosphatase showed Michaelis-Menten kinetics with its substrates. It had a Km of 1.5 microM and a Vmax of 3.2 mumol/min/mg with pyridoxal-P. The Vmax/Km was greatest with pyridoxal-P greater than 4-pyridoxic acid-P greater than pyridoxine-P greater than pyridoxamine-P. The phosphatase was competitively inhibited by the product, inorganic phosphate, with a Ki of 0.8 mM, and weakly inhibited by pyridoxal. It was also inhibited by Zn2+, fluoride, molybdate, and EDTA, but was not inhibited by levamisole, L-phenylalanine, or L(+)-tartrate. These properties of the purified enzyme suggest that it is a unique acid phosphatase that specifically dephosphorylates vitamin B6-phosphates.     

Relationship between phosphatase activity and cytotoxic effect of two protein phosphatase inhibitors, okadaic acid and pervanadate, on human myeloid leukemia cell line

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

Tartrate-inhibitable acid phosphatase. Purification from placenta, characterization and subcellular distribution in fibroblasts

Tartrate-inhibitable acid phosphatase was purified to apparent homogeneity from human placenta. The enzyme is composed of two subunits with an apparent molecular mass of 48 kDa. Each subunit carries one oligosaccharide of the high-mannose/hybride type. The purified enzyme has an isoelectric point of pH 6.2. It cleaves phosphomonoester bonds at acid pH, is competitively inhibited by L-tartrate, Ki = 0.51 microM, and phosphate, Ki = 0.8mM. A monospecific antiserum raised against the purified placental enzyme precipitated 62% and 85% of the tartrate-inhibitable acid phosphatase present in extracts of placenta and fibroblasts, respectively. By means of subcellular fractionation and immunoprecipitation it was shown that the majority of tartrate-inhibitable acid phosphatase is located in lysosomes in normal and mucolipidosis II fibroblasts. In the human Hep G-2 hepatoma cells a significant fraction of the enzyme appears to be associated with non-lysosomal organelles.     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.     

Cyclic nucleotide-independent phosphorylation of vitellin by casein kinase II purified from Rhodnius prolixus oocytes

In this study we show that Vitellin (VT) phosphorylation in chorionated oocytes of Rhodnius prolixus is completely inhibited by heparin (10 microg/ml), a classical casein kinase II (CK II) inhibitor. VT phosphorylation is not affected by modulators of cyclic nucleotide-dependent protein kinases such as c-AMP (10 microM), H-8 (1 microM) and H-89 (0.1 microM). We have obtained a 3000-fold VT-free enriched preparation of CK II. Autophosphorylation of this enzyme preparation in the presence of (32)P-ATP demonstrated that it lacks any endogenous substrates. Rhodnius CK II is strongly inhibited by heparin (Ki = 9 nM) and uses ATP (Km = 36 microM) or GTP (Km = 86 microM) as phosphate donors. Incubation of VT with purified Rhodnius CK II and (32)P-ATP led to the incorporation of 2 mols of phosphate/mol VT. However, the total number of phosphorylation sites available can be altered by previous incubation of VT with alkaline phosphatase. These data show that an insect yolk protein contain phosphorylation sites for a cyclic nucleotide-independent protein kinase such as CK II.     

Selective inactivation of an extra-cytoplasmic acid phosphatase of yeast-like cells of Sporothrix schenckii by sodium fluoride

Suspensions of intact, yeast-like cells of Sporothrix schenckii exhibited an acid phosphatase (EC 3.1.3.2) activity against p-nitrophenyl phosphate of about 5 IU (g dry wt)-1, without recourse to membrane perturbation. This extra-cytoplasmic acid phosphatase was reversibly and competitively inhibited by orthophosphate (Ki = 2 mM at pH 5) but unaffected by L(+)-tartrate (in contradistinction to some of the cytoplasmic acid phosphatases of the same organism). Inactivation by NaF of the extra-cytoplasmic isoenzyme was irreversible and followed first order kinetics; sensitivity to NaF was decreased by the presence of citrate, phosphate or substrate. Neither Km (0.3 mM at pH 5) nor Vmax for this enzyme in acetate buffer was greatly affected by pH in the range 3-5 but the first order rate constant for inactivation by NaF was strongly dependent on pH (maximum at pH 3.5). Crude cell-free extracts of yeast cells had nine electrophoretically distinct acid phosphatase activity bands and, on the basis of the pattern of inhibitors, the extra-cytoplasmic activity was identified as Y-I, an isoenzyme that barely penetrates standard polyacrylamide gel electropherograms. Additional evidence for the assignment came from selective inactivation of this isoenzyme by short treatments of intact cells with NaF under conditions that did not allow penetration of the plasma membrane by the inhibitor and did not kill the cells.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+

We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.     

Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties

The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Metabolism of 2-oxoaldehyde in mold. Purification and characterization of two methylglyoxal reductases from Aspergillus niger

Two kinds of methylglyoxal reductases were purified to apparent homogeneity from Aspergillus niger and designated MGR I and MGR II. Both enzymes consisted of a single polypeptide chain with a relative molecular mass of 36,000 (MGR I) and 38,000 (MGR II). NADPH was specifically required for the activities of both enzymes and Km values for NADPH were 54 microM (MGR I) and 6.8 microM (MGR II). MGR I was specific to 2-oxoaldehydes [glyoxal, methylglyoxal (Km = 15.4 mM) and phenylglyoxal], whereas MGR II was active on both 2-oxoaldehydes [glyoxal (Km = 10 mM), methylglyoxal (Km = 1.43 mM), phenylglyoxal (Km = 4.35 mM) and 4,5-dioxovalerate] and some aldehydes (propionaldehyde and acetaldehyde). Optimal pH values for MGR I and MGR II activities were 9.0 and 6.5 respectively. Both enzymes were inactivated by a brief incubation with 2-oxoaldehydes (glyoxal, methylglyoxal and phenylglyoxal) in the absence of NADPH. MGR I activity was competitively inhibited by NADP+ and the Ki value for NADP+ was calculated to be 0.49 mM. On the other hand, the inhibition of MGR II activity by NADP+ was of mixed type, the Ki value for NADP+ being 45 microM. MGR I was different from MGR II in amino acid composition.