Extracellular alkaline phosphatase from the filamentous fungusAspergillus caespitosus: Purification and biochemical characterization

Among 30 species of filamentous fungi isolated from Brazilian soil, Aspergillus caespitosus produced and secreted the highest levels of alkaline phosphatase in culture medium supplemented with xylan. The extracellular alkaline phosphatase was purified by DEAE-cellulose and concanavalin A-sepharose chromatography. The enzyme was a glycoprotein containing up to 56% sugar with molar mass of 134.8 kDa, according to gel filtration in Sepharose CL-6B, and 57 kDa according to SDS-PAGE. Nondenaturing electrophoresis (6% PAGE) of the purified enzyme produced a single band, suggesting that the native enzyme was a homodimer. Optima of temperature and pH were 75 degrees C and 8.5, respectively. The enzyme was stable at 50 degrees C and its activity was enhanced by 95% in the presence of Mg2+ (1 mmol/L). 4-Nitrophenyl phosphate was the preferentially hydrolyzed substrate with K(m) and upsilon lim values of 74 mumol/L and 285 mumol/s, in the absence, and 90 mumol/L and 418 mumol/s, in the presence of Mg2+, respectively. The enzyme also hydrolyzed other phosphorylated amino acids (O-phosphothreonine, O-phosphotyrosine, O-phosphoserine).     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

Purification and characterization of phytase from rat intestinal mucosa.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity.     

Mycelial forms of <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> present ectophosphatase activities

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.     

Structural and kinetic alterations of constitutive conidial alkaline phosphatase from the osmotically-sensitive mutant ofNeurospora crassa

The osmotically-sensitive os-1 mutant of Neurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 degrees C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate, O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by Co(II) (26%), Ni(II) (23%) and Mg(II) ions (80%). The enzyme was stable for up to 6 months at 4 degrees C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 degrees C for 10 d. The kinetic and structural properties of the conidial enzyme purified from the os-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

A green 2,4-pentadienoyl-CoA reductase from Clostridium aminovalericum

2,4-Pentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170-182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visible-spectrum showed absorption maxima at 270, 402 and 715 nm as well as shoulders at 278, 360, 450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two. The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoyl-CoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (V = 26 nkat/mg protein) or ferricenium hexafluorophosphate (V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA (V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA delta-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoyl-CoA.     

Alkaline phosphatase of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> is insoluble

The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg²⁺-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.     

Mechanism of action of Zn2+ and Mg2+ on rat placenta alkaline phosphatase. II. Studies on membrane-bound phosphatase in tissue sections and in whole placenta.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate

Polidocanol-solubilized alkaline phosphatase was purified to homogeneity with a specific activity of 822.3 U/mg. In the absence of Mg2+ and Ca2+ ions and at pH 9.4, the enzyme hydrolyzed ATP in a manner that could be represented by biphasic curves with V = 94.3 U/mg, K0.5 = 17.2 microM, and n = 1.8 and V = 430.3 U/mg, K0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity sites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the specific activity increased to as much as 1,000 U/mg, whereas the K0.5 and n values remained almost unchanged. In the presence of nonsaturating concentrations of metal ions, the hydrolysis of ATP was similar to that observed in the absence of these ions, but with a marked decrease in K0.5 values. At pH 7.5, the enzyme also hydrolyzed ATP with K0.5 = 8.1 microM and V = 719.8 U/mg. Apparently, alkaline phosphatase was able to hydrolyze ATP in vivo, either at pH 7.5 or pH 9.4. These data contribute to the knowledge of the biological properties of skeletal alkaline phosphatase and suggest that this enzyme may have a high-affinity binding site for ATP at alkaline pH.     

大豆磷酸烯醇式丙酮酸磷酸酯酶研究Ⅲ、非专一性酸性磷酸酯酶的纯化与特性

从大豆叶片中分离能水解磷酸烯醇式丙酮酸(PEP)的酸性磷酸酯酶,通过硫酸胺分部(20%～50%饱和度)沉淀、DEAE纤维素层析、刀豆球蛋白琼脂糖凝胶亲和层析将酶纯化了422.47倍,活性达78.16U/mg蛋白。该酶对PEP专一性不强,Km为1.09mmol/L(PEP),最适pH5.8,在pH4.8～7.0范围内及60℃以下较稳定,水解PEP的活性被Mg2+、Mn2+激活,F、Cu2+、Zn2+、PO43、MoO42及3磷酸甘油酸(3PGA)、三磷酸腺苷ATP等代谢物抑制,受异柠檬酸等有机酸影响较小。     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.     

树状多节孢酸性磷酸酶的分离纯化与性质研究

树状多节孢Nodulisporium sylviforme是从东北红豆杉Taxus cuspidata分离、可产生紫杉醇的内生真菌。研究以树状多节孢为材料,利用液体发酵手段获得菌丝体,通过CM-cellulose阴离子交换柱层析、Q-Sepharose阳离子交换柱层析和FPLC凝胶过滤层析（Superdex 75）,获得纯化的树状多节孢酸性磷酸酶蛋白（Nod-ACP）。结合FPLC和SDS-PAGE分析,判定该磷酸酶为分子量44kDa单亚基蛋白。酶学性质研究表明,其最适pH值为3.0,最适温度为58℃。6     

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).     

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain.

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.     

Activation of bovine heart ATP-MG2+-dependent phosphoprotein phosphatase: isolation of a phosphoenzyme intermediate and its conversion to the active form via a Mg2+-dependent autodephosphorylation reaction

The ATP-Mg2+-dependent protein phosphatase, a holoenzyme form of type I protein phosphatase (phosphatase-1) requires the action of phosphatase-1 kinase (FA) for activation. The enzyme (75 kDa) purified from bovine heart consists of a catalytic (C) and a regulatory (R) subunit of 40 kDa and 34 kDa, respectively, and activation is associated with phosphorylation of the R-sub-unit. A procedure has been developed for isolation of [32P]phosphatase-1 ( [32P]E-P) in non-denatured form. In the absence of divalent cation, [32P]E-P is catalytically inactive and the phosphorylation is stable. Addition of Mg2+ triggers autodephosphorylation of [32P]E-P with concomitant generation of phosphorylase phosphatase activity. The autodephosphorylation/activation process is dependent on Mg2+ concentration. The KA value for Mg2+ is 0.6 mM. The phosphorylase phosphatase activity generated from the release of 1 mol. 32P is 1.1 X 10(12) units which is equivalent to 15,000 units per mg enzyme protein. The present findings provide direct evidence that the phosphorylated phosphatase-1 is not the active form (Ea). Instead, Ea is directly produced from the intermediate by a Mg2+-dependent autodephosphorylation reaction.