Interaction of the 56,000-dalton phosphoprotein phosphatase from reticulocytes with regulin and inhibitor 2

The interaction of divalent metal ions with a homogeneous 56,000-dalton phosphoprotein phosphatase isolated from rabbit reticulocytes was studied. The effects of the ions on enzymatic activity and on fluorescence from a 3-(4-maleimidylphenyl)-4-methyl-7-(diethylamino)coumarin derivative of the protein were compared. Enzymatic activity is dependent on Mn2+. The apparent association constant for Mn2+ is about 0.5 mM-1 as judged from enzymatic activity and from changes in fluorescence caused by binding of the metal ion; Ca2+ and Mg2+ do not affect enzymatic activity and appear not to bind tightly to the enzyme; however, Co2+, Fe2+, and Zn2+ bind to the protein and inhibit the Mn2+-activated enzyme. The 56,000-dalton phosphoprotein phosphatase was found to interact with regulin, a spectrin-associated protein also isolated from reticulocytes, and with skeletal muscle phosphatase inhibitor 2. The interaction was followed by changes in the enzymatic activity and by quenching of fluorescence from the coumarin derivative of the phosphatase. Homogeneous regulin (Mr approximately 230,000) increases the activity of the enzyme severalfold; this stimulation is Mn2+-dependent. Inhibitor 2 decreases enzyme activity but only if the two proteins are preincubated in the absence of Mn2+. Comparable differences in the effect of Mn2+ were also observed in parallel experiments in which changes in fluorescence from the coumarin-labeled 56,000-dalton phosphatase were measured. In these experiments, it was shown that Mn2+ enhances the interaction between regulin and the 56,000-dalton phosphatase, but inhibits the interaction between the phosphatase and inhibitor 2.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Purification of Choline Acetyltransferase from the Locust Schistocerca gregaria and Production of Serum Antibodies to This Enzyme

Choline acetyltransferase (ChAT; EC 2.3.1.6) was purified from the heads of Schistocerca gregaria to a final specific activity of 1.61 mumol acetylcholine (ACh) formed min-1 mg-1 protein. The molecular mass of the enzyme as determined by gel filtration is 66,800 daltons. The final enzyme preparation showed one major band at 65,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which corresponds with the native molecular mass of the enzyme, a band at 56,000 daltons, and two bands at 40,500 and 38,000 daltons. Antibodies raised against ChAT in rabbit react only with the active band on native gel after Western blotting. They strongly react with the 65,000-dalton polypeptide band on Western blots of SDS gel separation of pure preparation of enzyme and with both the 65,000- and 56,000-dalton bands after SDS gel separation of crude extract.     

Purification to homogeneity and partial characterization of a 56,000-dalton protein phosphatase from rabbit reticulocytes

A 56,000-Da peptide with inherent protein phosphatase activity was isolated from the postribosomal supernatant fraction of rabbit reticulocytes. The peptide appears to form complexes with other proteins that are present in crude fractions. It exhibits atypical retention on steric exclusion columns during high performance liquid chromatography, an unusual characteristic that facilitated its isolation. The protein phosphatase activity of the 56,000-Da peptide is dependent on Mn2+ ions, but is not activated by either the FA, ATP/Mg2+ protein phosphatase activator system or by proteolysis. The protein phosphatase activity of the peptide is increased 3-fold or more by the antigen peptides described in the accompanying paper (Fullilove, S., Wollny, E., Stearns, G., Chen, S.C., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2493-2500).     

Evidence for a proteolytic modification of liver pyruvate kinase in fasted rats.

Liver pyruvate kinase was purified to homogeneity from rats fed a high carbohydrate, low protein diet (LPK-C) and from rats fasted for 84 h (LPK-F). Although the enzymes have similar electrophoretic mobilities in 7% polyacrylamide disc gels, the specific activity of LPK-C was two to three times the value of the specific activity of LPK-F. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPK-C yields a single protein band of 56,000 daltons. In contrast, LPK-F yields two bands of protein. Approximately one-third of the LPK-F has an electrophoretic mobility similar to the 56,000-dalton LPK-C peptide. The remaining two-thirds of the LPK-F protein migrates as a 51,000-dalton peptide. Cyanogen bromide was used to cleave LPK-C and LPK-F. Similar peptide patterns were obtained from LPK-C and LPK-F when the cyanogen bromide fragments were resolved by 12% polyacrylamide gel electrophoresis in 7.5 m urea containing 6 mm Triton X-100 and 5% acetic acid. Separation of the two peptides from LPK-F was accomplished by selective immunologie absorption of the 56,000-dalton peptide with anti-LPK-C gammaglobulin immobilized on Sepharose 4B. Tryptic digests of LPK-C, LPK-F and the 51,000-dalton peptide yield similar peptide patterns when analyzed via sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. These results suggest that the 51,000-dalton peptide could be derived by a proteolytic cleavage or limited digestion of the 56,000-dalton subunit. Phosphorylation of LPK-C and LPK-F by [γ-³²P]ATP in vitro with cyclic AMP-activated protein kinase results in covalent incorporation of ${}^{32}$P into only the 56,000-dalton subunit. These results suggest that anin vivo proteolytic modification that yields the 51,000-dalton subunit.     

Characterization of a calmodulin-dependent protein phosphatase from human platelets

A calmodulin-dependent protein phosphatase has been identified in human platelets by its cross-reactivity with an antibody developed against a bovine brain calmodulin-dependent protein phosphatase and by its calmodulin-stimulated dephosphorylation of 32P-labeled substrates. The platelet enzyme was partially purified to separate it from calmodulin and calmodulin-independent phosphatases. The partially purified enzyme was stimulated by calmodulin, requiring 15 nM calmodulin for half-maximal activation. Calmodulin increased the Vmax of the phosphatase, with no significant effect on its Km. The enzyme was stimulated irreversibly and made calmodulin-independent by limited proteolysis. The optimal pH for the phosphatase was 7.5. After partial purification, phosphatase activity was significantly increased in the presence of Mn2+ and Ca2+ over that observed in the presence of Ca2+ alone. The enzyme effectively dephosphorylated casein, histone, protamine, and platelet actin. The holophosphatase was estimated to have a molecular weight of 76,900 as determined by sedimentation on sucrose gradients. Immunoblotting techniques using an antibody against the brain phosphatase suggests that the enzyme consists of 2 subunits of 60,000 and 16,500 daltons; the 60,000-dalton subunit co-migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 60,000-dalton calmodulin-binding protein in the platelet suggesting that it is the calmodulin-binding subunit of the enzyme. The identification of a calmodulin-dependent protein phosphatase in human platelets suggests a role for Ca2+-dependent dephosphorylation in platelet activation.     

Cobalt-dependent protein phosphatases from human cord blood erythrocytes. I. Submolecular structure and regulation of activity of E3 casein phosphatase

We have identified three phosphoprotein phosphatases in the cytosol of human cord blood erythrocytes by sequential anion-exchange chromatography and gel filtration. The most abundant was E3 protein phosphatase. After rechromatography on a column of Ultrogel AcA-44 the enzyme had a molecular weight of 95,000 daltons. According to the data obtained by SDS/PAGE, the 95,000-dalton form was composed of non-identical subunits with a molecular mass of 23,000 and 16,000 daltons. Since ethanol decreased the molecular mass of the 95,000-dalton enzyme to 25,000 daltons, we suggest that the protein of 23,000-25,000 daltons represents the catalytic subunit. The decrease in the molecular weight is followed by a 2-fold increase in the Vmax value and by a change in kinetics: the negatively cooperative 95,000-dalton enzyme (h = 0.45) transforms into Michaelis-Menten kinetics (h = 1.0) in the 25,000-dalton form. Both molecular forms, 95,000 and 25,000 daltons, only dephosphorylated casein but not phosvitine and histones. Both forms were activated by CoCl2 and inhibited by organic, and most potently, by inorganic pyrophosphates to approximately the same degree. As opposed to the inorganic pyrophosphate, which affects the catalytic properties of the enzyme molecule, CoCl2 did not affect the catalytic properties of the enzymes, but it probably did affect the rate of 'E-S' complex formation. CoCl2 protected the 95,000-dalton enzyme from pyrophosphate inhibition. The data indicate that CoCl2 and pyrophosphate may take part in the regulation of the activity of both forms of E3 phosphatase.     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

Cobalt-dependent protein phosphatases from human cord blood erythrocytes. II. Further characterization of E2 casein phosphatase

Of three casein phosphatases isolated from the cytosol of human cord blood erythrocytes two were cobalt-dependent, E2 and E3. In the presence of CoCl2, E2 activity was the most prominent. In addition to casein, E2 dephosphorylated phosvitin and p-nitrophenyl phosphate (p-NPP) with pH optima at 6.8-7.2 for proteins and 9.0 for p-NPP. The native enzyme had a molecular weight of 104,000 daltons after AcA-44 Ultrogel filtration. According to SDS/PAGE it consisted of two subunits, 78,000 and 15,000 daltons. The 104,000-dalton form exhibited Michaelis-Menten kinetics and had the greatest affinity for casein between protein substrates tested. Ethanol denaturated the enzyme by 80%. Optimal activation of E2 phosphatase was achieved with 5 mmol/l CoCl2 which did not affect the catalytic properties of the enzyme but did affect the rate of 'E-S' complex formation. Inorganic pyrophosphate was not inhibitory for the 104,000-dalton enzyme. Judging by all these properties the natural substrate for E2 casein phosphatase could be P-pyruvate kinase.     

Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase

Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, whereas similar treatment of sparse cells causes only a marginal increase in cyclic AMP formation. To determine which membrane protein(s) is altered under conditions which result in proteolytic activation of adenylate cyclase, purified plasma membranes and intact normal rat kidney cells were subjected to limited proteolysis and membrane proteins analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Membranes prepared from intact confluent normal rat kidney cells exposed to mild trypsinization showed a decrease in proteins of 56,000, 46,000, 37,000, and 32,000 daltons. Trypsin treatment of intact, sparse cells does not activate the cyclase system and does not lead to modification of the 46,000-dalton membrane protein. Treatment of purified normal rat kidney plasma membranes results in the loss of numerous bands in the high molecular mass region (>150,000 daltons) as well as decreases membrane proteins of 56,000, 49,000, 46,000, and 23,000 daltons. Compared with trypsin, the proteolytic action of papain appears to be quite specific, causing a discernible decrease in only the 46,000-dalton protein. The correlation between modification of the 46,000-dalton membrane component and the activation of the cyclase system suggests that perhaps this protein is proteolytically modified to elicit activation of adenylate cyclase.     

Purification of a myosin phosphatase from bovine aortic smooth muscle

A myosin phosphatase has been purified to homogeneity from bovine aortic smooth muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme eluted from nondenaturing gels revealed two subunits (Mr = 67,000 and 38,000). Densitometric scans of the subunits indicated a molar ratio of 1:1. Several phosphoproteins were substrates for the phosphatase including histone II-A, isolated 20,000-dalton smooth muscle myosin light chains, phosphorylase a, and smooth muscle myosin. In the presence of 0.25 M NaCl and a substrate concentration of 2 microM, myosin was preferentially dephosphorylated. The specific activity of the phosphatase for myosin at a concentration of 10 microM was found to be 5 mumol/mg/min. The phosphatase required Mn2+ or Co2+ ions for activity. Mg2+, Ca2+, or Mg-ATP would not substitute for Mn2+ or Co2+ at equimolar concentrations. This phosphatase may play an important role in regulating actin-myosin interaction in smooth muscle by serving to dephosphorylate myosin.     

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.     

Partial purification and characterization of reticulocyte phosphatase with activity for phosphorylated peptide initiation factor 2

An enzyme fraction containing phosphatase activity for phosphorylated eukaryotic peptide initiation factor 2 (eIF-2) has been isolated from rabbit reticulocytes and partially characterized. The enzyme efficiently catalyzes release of phosphate from the small subunit of eIF-2 (eIF-2 alpha) that has been phosphorylated by the hemin-controlled repressor. It is shown to restore activity of this phosphorylated eIF-2 for binding of methionyl-tRNAf to 40 S ribosomal subunits in a partial reaction of peptide initiation. The enzyme fraction also has phosphatase activity for eIF-2 phosphorylated in its largest subunit and for the 100,000-dalton peptide associated with the eIF-2 alpha kinase activity of the hemin-controlled repressor. The phosphoprotein phosphatase has been isolated by a procedure involving precipitation with ethanol at room temperature and has an apparent molecular weight in the order of 76,000. Its phosphatase activity for eIF-2 alpha is stimulated about 3-fold by optimal concentrations of Mn2+, but is not stimulated by Ca2+ or Mg2+. The enzyme is strongly inhibited by Fe2+ and by purine nucleoside diphosphates.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Assignment of the alpha and beta chains of human propionyl-CoA carboxylase to genetic complementation groups.

Propionicacidemia is a metabolic disorder resulting from a deficiency of propionyl-CoA carboxylase activity. The enzyme is composed of two polypeptides: a 72,000-dalton alpha chain which contains the biotin ligand and a 56,000-dalton beta chain. It has been suggested that the two major complementation groups in this disorder, pccA and pccBC (with subgroups pccB and pccC), correspond to the genes encoding these two chains. To correlate gene product with complementation groups, 15 mutant and four normal human fibroblast strains were analyzed by [35S]methionine and [3H]biotin labeling. Immunoprecipitation and gel electrophoresis of the polypeptides revealed that alpha chains are synthesized by mutants of pccBC and both subgroups but not in four out of five pccA mutants. On the other hand, beta chains were detected only in pccB mutants. We suggest that pccA encodes the alpha chain of PCC while pccBC encodes the beta chain, and furthermore predict that the beta chain is unstable in the absence of the alpha chain.     

Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes.

Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given.     

Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown $\text{Hydrogenomonas eutropha}$ can be dissociated with sodium dodecylsulfate into small 15,000-dalton subunits and large 56,000- and 52,000-dalton subunits (the latter in a mole ratio of 5:3). The overal mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.     

Purification and properties of eIF-2 phosphatase

Eukaryotic initiation factor 2 (eIF-2) phosphatase has been purified 840-fold to apparent homogeneity from rabbit reticulocyte lysate. Native eIF-2 phosphatase has a Mr = 98,000, pI = 6.1, s20,w = 5.1, and a Stokes radius = 38 A. A subunit composition of one 60,000-dalton polypeptide and one 38,000-dalton polypeptide is indicated. The Km for [32P]eIF-2 is 30 microM and the Vmax = 1.1 nmol of phosphate released/min/microgram. The 38,000-dalton subunit of eIF-2 phosphatase does not co-electrophorese with the catalytic subunit of liver phosphorylase phosphatase, a type 1 protein phosphatase. The specificity of eIF-2 phosphatase for phosphorylation sites on th alpha- and beta-subunits of eIF-2 appears to be determined by the environment of the phosphatase and substrate. Both the alpha- and beta-subunits of [32P]eIF-2 are rapidly dephosphorylated by the purified phosphatase. In unfractionated lysate and in unfractionated lysate supplemented with an equivalent activity of the purified phosphatase, only the alpha-subunit of eIF-2 is dephosphorylated. This indicates other factors are present in the lysate which govern the dephosphorylation of eIF-2.