Heng-Chun Li

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.

Multiple forms of phosphotyrosyl- and phosphoseryl-protein phosphatase from cardiac muscle: Partial purification and characterization of an EDTA-stimulated phosphotyrosyl-protein phosphatase

Chromatography of cardiac muscle and brain extracts on DEAE-cellulose resolved phosphotyrosyl-protein phosphatase activity into three fractions, termed Y-1, Y-2, and Y-3. These were eluted at 0.05, 0.15, and 0.3 M KCl, representing about 33, 55, and 12%, respectively, of the enzymatic activity recovered from the resin. Comparative studies demonstrated that the properties of phosphatases Y-1, Y-2, and Y-3 were distinctly different from those of previously identified phosphoseryl-protein phosphatases-1, -2, -3, and -4. Phosphatases Y-1, Y-2, and Y-3 were stimulated by EDTA, and exhibited optimal activity at neutral pH. These properties were different from those of the two minor phosphotyrosyl-protein phosphatase activities associated with phosphoseryl-protein phosphatases-3, and -4, which were divalent cation dependent, and exhibited optimal activity at alkaline pH. Further purification of phosphatase Y-2 from bovine heart has been carried out. The enzyme had a Mr = 65,000 (Stokes radius = 3.8 nm; S020,W = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA (Ka = 15 microM) and was strongly inhibited by micromolar concentrations of vanadate. Phosphatase Y-2 was highly specific for phosphotyrosyl-IgG and -casein, and showed little activity toward phosphoseryl-casein, -phosphorylase a, phosphothreonyl-inhibitor-1, and p-nitrophenyl phosphate. The present studies indicate that phosphotyrosyl-protein phosphatase activity in animal tissues exists in multiple forms. The major active species are specific for phosphotyrosyl proteins, and represent enzymes different from the known phosphoseryl-protein phosphatases and p-nitrophenyl phosphatases.

Orthophosphate and histone dependent polyphosphate kinase from E.coli

Abstract A polyphosphate kinase has been purified over 100-fold from an extract of E. coli K-12. It requires both orthophosphate and a basic protein (histone or protamine) for maximum activity. Because its activity is stimulated by histone, polyphosphate kinase may easily lead to an error in the determination of protein kinase in the cell extract. Our data suggest that the stimulatory effect of orthophosphate on polyphosphate kinase may be important in the regulation of phosphate metabolism in the microorganism.

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

Abstract 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; Mg 2+ 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.

Isolation and properties of a Mn2+-activated phosphohistone phosphatase from canine heart

Abstract A Mn 2+ -activated phosphohistone phosphatase has been isolated from canine heart. The s 20, w for the enzyme is 3.8. Using this value and the value for Stokes radius (39 A), the molecular weight for the enzyme was calculated to be 61,000. The enzyme is inactive in the absence of divalent cations, among which Mn 2+ is the most effective activator. Co 2+ and Mg 2+ are less effective than is Mn 2+ . Zn 2+ , Fe 2+ , and Cu 2+ are inhibitory. The enzyme has a pH optimum between 7 and 7.5 and has an apparent K m for phosphohistone and Mn 2+ of about 17 μ m and 0.5 m m , respectively. The enzyme is inhibited by nucleoside triphosphate, ADP, AMP, phosphate, and pyrophosphate, but is not affected by cyclic AMP or cyclic GMP. The dephosphorylation of phosphohistone is stimulated by salts. Kinetic studies reveal that KCl and other salts greatly affect both the rate of hydrolysis and the K m for either Mn 2+ or phosphohistone by interacting with the substrate. The data suggest that modification at substrate level is an important regulatory mechanism for the enzyme. The enzyme preparation also dephosphorylates phosphorylase a and phosphocasein. Evidence suggests that one enzyme possesses both phosphohistone and phosphorylase phosphatase activities and that a different enzyme catalyzes the Mg 2+ - and Mn 2+ -activated dephosphorylation of phosphocasein.

A novel glycogen synthase phosphatase from canine heart

Summary Two molecular forms (M r =49,300 and 26,000) of a divalent cation-dependent (Mg 2+ =Mn 2+ >Co 2+ ) protein phosphatase, which represent the major glycogen synthase phosphatase activity in canine heart extracts, have been partially purified and characterized. Although a general protein phosphatase of M r =35,000 is also active toward synthase D, it represents the major phosphorylase phosphatase activity in heart muscle. The present findings indicate that the dephosphorylation of synthase D and phosphorylase a may be regulated by two distinct phosphatases rather than by a single nonspecific enzyme.

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.

Dethiophosphorylation of thiophosphorylase a by a multifunctional phosphoprotein phosphatase of Mr=35,000☆

Abstract It is generally believed that proteins thiophosphorylated by adenosine 5′-0-(3-thiotriphosphate) (ATPγS) are resistant to phosphoprotein phosphatases. We have found that a general phosphoprotein phosphatase of Mr=35,000 purified from bovine heart, rabbit muscle or liver, can efficiently catalyze the dethiophosphorylation of [ 35 S ]thiophosphorylated phosphorylase ([ 35 S ]thiophosphorylase a ) . The rate of the dethiophosphorylation reaction in the presence of lmM Mn 2+ was about one-third as that of dephosphorylation of [ 32 P ]phosphorylase a . The dethiophosphorylation reaction was almost completely inhibited by EDTA and was stimulated several fold by Mn 2+ . On the other hand, the dephosphorylation of [ 32 P ]phosphorylase a was not significantly affected by these reagents. The present findings indicate that thiophosphorylated proteins in general may not be resistant to phosphoprotein phosphatases.