Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Serine transhydroxymethylase isoenzymes from a facultative methylotroph.

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.     

Isolation and some properties of phospholipase D from Bacillus subtilis G-22]

A method of isolating highly purified phospholipase D from Bac. subtilis G-22 is described. It includes ammonium sulphate fractionation, thermal denaturation, chromatography on lipoprotein bound with sepharose 6B and AH-sepharose 4B. The enzyme is 130-fold purified, its yield exceeds 90.0%, its specific activity is 164 units per mg of protein. The homogeneity of the enzyme is demonstrated by polyacrylamide gel electrophoresis, ultracentrifugation, isoelectric focusing and N-terminal amino acid determination by means of dinitrophenylation and dancylation. Proline is found to be N-terminal amino acid. The molecular weight of the enzyme, as determined from gel filtration through Sephadex G-100, is 21500 +/- 300, its sedimentation constant is 1.4S, isoelectric point is at pH 4.2. The molecular weight calculated from amino acid composition, is 21000--22000. Polypeptide chain contains of 196--205 amino acid residues. Phospholipase D develops its maximal activity at pH 8.5 and does not contain free SH-groups. Benzylsulphofluoride does not inhibit the enzyme activity. Phospholipase D is activated by Cd2+, Co2+, Zn2+, Ca2+ and is inhibited by EDTA, pIi50 being about 2.6.     

RNase E, an RNA processing enzyme from Escherichia coli.

An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.     

Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius.

D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-CO2-S2O3(2)-grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 +/- 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 +/- 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The S(0)20,w of the enzyme was 18.07S +/- 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 mumol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3-.     

Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.     

Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate

4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.     

Purification and properties of a novel nucleolar exoribonuclease capable of degrading both single-stranded and double-stranded RNA

A ribonuclease that hydrolyzes either linear duplex or single-stranded RNA in an exonucleolytic manner has been partially purified from Ehrlich ascites tumor cell nucleoli and is free from other ribonucleases. The enzyme will also degrade the RNA complement of an RNA X DNA duplex; however, no nuclease activity is observed on linear duplex or single-stranded DNA. The exonuclease acts on RNA nonprocessively from the 3' end releasing 5'-mononucleotides. The enzyme has a broad pH optimum around pH 8.0, requires Mg2+ or Mn2+ (0.06 mM) for optimum activity, and is sensitive to ethylenediaminetetraacetic acid and N-ethylmaleimide inhibition. Monovalent cations including K+, Na+, and NH4+ are inhibitory. Gel filtration studies of this enzyme gave a Stokes radius of 40 A. Sedimentation velocity measurements in glycerol gradients yield a S20,W of 6.0 S. From these values a native molecular weight of 100 000 was calculated. Copurification of the single- and double-stranded activities, identical reaction requirements, and identical heat-inactivation curves strongly suggest that both activities reside with the same enzyme.     

棒曲霉Asp-195v产蛋白酶的酶学性质研究

对液体发酵的棒曲霉Asp-195v菌株所产蛋白酶的活力进行了研究,并通过分离纯化获得了电泳纯的酶蛋白。研究结果表明,该蛋白酶的最适反应温度为40℃,在30-50℃温度范围内相对活力可保持在70%以上;最适pH为7,pH稳定范围在4-8;Mn2+对该蛋白酶活力有明显的激活作用,K+、Ag+、Cu2+、Fe2+、Mg2+、Zn2+、Ca2+、Al3+和Fe3+离子则有明显的抑制作用,尤其是Hg2+和Pb2+对酶活的抑制作用更加强烈;其他试剂如葡萄糖、EDTA对酶活的抑制作用不明显,而蔗糖、SDS和Tween-20对酶活的抑制明显;以酪氨酸为底物采用双倒数作图法测得Vmax为30.40mmol/min,Km为97.53mmol/L。该酶的表观分子量为30.1kDa。     

几丁质酶产生菌筛选鉴定及产酶性能研究

从土壤样品中筛选得到一株高产几丁质酶菌株C65-2,经形态学观察和18S rDNA序列测定,鉴定为Aspergillus fumigatus,对产酶培养基进行初步优化,测得最高酶活可达6.9U/ml,酶活力较优化之前提高了210%。酶学性质研究表明该几丁质酶分子量约为20kDa,酶在60℃下保温50min酶活降为0,最适酶反应温度是55℃,酶反应最适pH为7.0,Mg2+,Cu2+对酶反应有促进作用,Fe3+对酶反应有抑制作用。     

Purification and characterization of a neutral protease from rat-liver cytosolic fraction

Rat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metal-depleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200,000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110,000, 74,000, and 40,000.     

Purification and properties of methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns. The molecular weight, estimated by gel electrophoresis, was found to be 122,000, and the enzyme contained two different subunits with molecular weights of 34,000 and 53,000, which indicates an alpha 2 beta structure. The enzyme contains three or four molecules of 5-hydroxybenzimidazolylcobamide, which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate. In both cases the enzyme dissociated into its subunits. For stability, the enzyme required the presence of divalent cations such as Mg2+, Mn2+, Sr2+, Ca2+, or Ba2+. ATP, GTP, or CTP was needed in a reductive activation process of the enzyme. This activation was brought about by a mixture of H2, ferredoxin, and hydrogenase, but also by CO, which is thought to reduce the corrinoid chemically. The CO dehydrogenase-like activity of the methyltransferase is discussed.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.     

Purification, some properties and quaternary structure of the D-ribulose 1,5-diphosphate carboxylase of Alcaligenes eutrophus.

D-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacterium Alcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000. Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2. Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2+, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.     

Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

嗜热真菌Thermomyces lanuginosus A_(236)热稳定葡萄糖淀粉酶的纯化及其特性

嗜热真菌ThermomyceslanuginosusA_236在液体培养基中50℃下静止培养14天,粗提酶液经硫酸铵分级沉淀、DEAE-Toyopearl离子交换层析、Butyl－Toyopearl疏水层析、SephacrylS100凝胶过滤和FPLCMonoQ离子交换层析,得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经TLC分析为葡萄糖,证明纯化的酶为葡萄糖淀粉酶（EC3．2．1．3）。SDS－PAGE测定其分子量为72,000,不具亚基,PI为4．0,富含Val和Leu。酶反应最适温度和pH分别为70℃和5．0。在pH5．0条件下,酶在60℃保温1h,仍具有原酶活性。酶活性在70℃和80℃的半衰期分别为20min和6min。Ca2+对酶有激活作用,Fe3+、Al3+、Hg2+等金属离子对酶活力有一定的抑制作用。纯酶碳水化合物含量为12．4％。纯酶可水解可溶性淀粉、直链淀粉、支链淀粉、糊精、糖原、麦芽三糖和麦芽糖,其中可溶性淀粉为最适底物。     

Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.     

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.