Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Comparison of the liver glycogen synthase from normal and streptozotocin-induced diabetic rats

Glycogen synthase in the liver extracts of short-term (3 days) streptozotocin-induced diabetic rats is poorly activated by the endogenous synthase phosphatase as well as phosphatase(s) from the liver extracts of normal animals. However, synthase in the liver extracts of diabetic rats is readily activated by the 35,000 M_r rabbit liver protein phosphatase (H. Brandt, F. L. Capulong, and E. Y. C. Lee (J. Biol. Chem.250, 8038–8044 (1975)). The purified synthases from normal and diabetic animals respond differently after incubations with three different phosphatases. Both normal and diabetic synthase are activated by the 35,000 M_r protein phosphatase; however, the total activity of diabetic, but not the normal, synthase is significantly increased. Normal, but not the diabetic, synthase is activated by a synthase phosphatase from normal rats; this activation is accompanied by an increase in total synthase activity. Incubation of the diabetic synthase with calf intestinal alkaline phosphatase results in a reduction of the total synthase activity, whereas synthase activity of the normal is not significantly affected. The reduction in total activity of the diabetic synthase by treatment with alkaline phosphatase was prevented by prior dephosphorylation with 35,000 M_r rabbit liver protein phosphatase. In addition to their differences in responses to different phosphatases, the normal and diabetic synthases are also different in their molecular weights as determined by sucrose density gradient centrifugation (154,000 ± 17,000 (n = 6) for the normal and 185,000 ± 15,000 (n = 8) for the diabetic synthase) and their kinetic properties.     

Identification and partial characterization of bovine heart cytosolic phosphorylase phosphatases

The properties of phosphatases in bovine heart cytosol were studied. Two isozymic forms of protein phosphatase H (H-1 and H-2) were resolved by chromatography on DEAE-Sephacel. The two isoenzymes had identical physical properties (Mr 260,000, 7.9 S). Treatment with 80% ethanol activated both isozymes and converted H-1 to a Mr 35,500 form and H-2 to Mr 67,000 and Mr 35,500 forms. Both H-1 and H-2 and their lower Mr activated forms had essentially identical Km values for phosphorylase a. The heart cytosol also contained a latent phosphatase (Fc) which could be activated by preincubation with either ATP X Mg and an activating factor (FA), or by Mn/trypsin treatment. The latter procedure converted the latent Fc (Mr 200,000) to a Mn2+-independent Mr 34,500 form. Both activated forms of Fc had similar Km values which were fourfold lower than the affinity of the protein phosphatase H forms for the phosphorylase a substrate.     

The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high $\text{pH}\text{6.8}\text{pH}\text{8.2}$ activity ratio (0.4 – 0.7) and a high level of Ca²⁺ independent activity $\text{(EGTA}\text{Ca}{}^{\mathrm{2+}}\text{= 0.3?0.5)}$. The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a M_r 128,000 polypeptide in the gradients. This polypeptide and a M_r 143,000 polypeptide were labeled with ³²P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a M_r 102,000 membrane polypeptide which followed the distribution of Ca²⁺-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A M_r 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca²⁺/calmodulin-stimulated manner, including a M_r ca. 72,000 polypeptide found only in the transverse tubule.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

Oligomeric forms of plant acetolactate synthase depend on flavin adenine dinucleotide

Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (M_r = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (M_r = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directly-formed acetoin indicate similar properties of these separated ALS forms.     

Intact form of myeloperoxidase from normal human neutrophils

Myeloperoxidase (donor: H₂O₂ oxidoreductase, EC 1.11.1.7) of human polymorphonuclear neutrophils was purified rapidly in the presence of the protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The purified enzyme behaved as a single molecular species in several nondenaturing electrophoretic and chromatographic systems. Peroxidase activity in fresh extracts of neutrophils from 20 normal persons and from 5 patients with polycythemia was electrophoretically identical to purified enzyme. Treatment with trypsin converted myeloperoxidase to multiple electrophoretic forms of active enzyme. Size (M_r ca. 15,000 and ca. 55,000) and stoichiometry of the subunits of purified enzyme, and enzyme M_r ca. 140,000, were compatible with intact myeloperoxidase having an α₂β₂ structure. We found no evidence for electrophoretically detectable genetic polymorphism of myeloperoxidase. Proteolytic degradation of myeloperoxidase probably accounted for electrophoretic heterogeneity of enzyme and for some constituent peptides described previously.     

Release and activation of phosphorylase phosphatase upon rupture of organelles from rat liver

Liver extracts (8000 × $\text{g}$ for 10 min) from fasted rats contain about 4 times more phosphorylase phosphatase activity when the liver was homogenized in a hypotonic medium or frozen before homogenization. This increase is caused by: (i) release of partially latent phosphatases ($\text{M}$_r=60 000 and 45 000 in sucrose gradient centrifugation) from ruptured organelles; (ii) rapid activation of phosphatase in the ruptured pellet by endogenous protease(s) which can be blocked by p-tosyl-L-lysine chloromethyl ketone. Only the $\text{M}$_r=60 000 enzyme, associated with the nuclei, can be activated proteolytically, with conversion to an $\text{M}$_r=45 000.     

Purification and properties of bovine myocardial phosphorylase phosphatase (protein phosphatase C).

Phosphorylase phosphatase was purified to homogeneity from bovine myocardium by the procedure of Brandt et al. (Brandt, H., Capulong, Z. L., and Lee, E. Y. C., (1975) J. Biol. Chem.250, 8038–8044). The purified enzyme consists of a single polypeptide chain of M_r, 34,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The K_m for phosphorylase a was 2.9 μm and at V_max, the enzyme had a turnover number of 11.7 mol phosphorylase a (dimer) converted/mol phosphatase/s. Phosphorylated histone and protamine were also substrates for this phosphatase. The K_m for histone was 46 μm (based on incorporated ³²P_i and at V_max a turnover number of 3.3 mol phosphate released/mol phosphatase/s was found. In general, the properties of the bovine phosphorylase phosphatase closely resemble those found for the rabbit liver enzyme.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

Kinetics of photosynthetic membrane protein assembly in Rhodopseudomonas spheroides

Glycogen phosphorylase in cell-free extracts of Neurospora crassa is activated 10- to 15-fold by incubation with MgATP^2?. When the MgATP^2? is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg²⁺ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-1-phosphate and a linear non-competitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to 3-fold by 5′-AMP. As the b form is purified, the 5′-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.     

Chromatographic characteristics and subcellular localization of synthase phosphatase,phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes

Summary Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation charcteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75–85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100.When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained.Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 × g supernatant.Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a M_r 30 000 species. Heat treatment of the M_r 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity.Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was notinhibited by synthase D (8.5 unit/ ml) orby phosphorylase a(12 unit/ ml).     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.     

Polypeptide structure of human terminal transferase

The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of M_r 58-56,000 and M_r 44-42,000 are the major immunoreactive polypeptides. Only the M_r 44-42,000 polypeptides can be efficiently renatured $\text{in}\text{situ}$ after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the M_r 58,000 polypeptide.     

Inhibitor-1 phosphatase activity in vascular smooth muscle

The histone-H1 and polylysine stimulated "latent" phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with beta-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.