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).     

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver.

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrates purified liver synthase D, phosphorylase alpha and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10 fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphorylase phosphatase by synthase D (Ki approximately 2 units/ml). The inhibition of synthase phosphatase by phosphorylase alpha was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase alpha. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase alpha and histone in the cell.     

Changes in receptor activator of nuclear factor-kappaB, and its ligand, osteoprotegerin, bone-type alkaline phosphatase, and tartrate-resistant acid phosphatase in ovariectomized rats

We investigated time-course changes in the expression of receptor activator of nuclear factor-kappaB (RANK), its ligand (RANKL), osteoprotegerin (OPG), bone-type alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRAP) in ovariectomized (OVX) rats. Samples of sera and coccyges were used for analysis of the enzyme activities and expression levels of proteins and mRNAs, and an immunohistochemical analysis was also performed. Serum BAP activity increased to 158.6% of the pre-operation value at 1 week after OVX, and then decreased to 38.7% at 8 weeks after OVX. On the other hand, the serum TRAP activity increased to 130.9% of the pre-operation level at 1 week after OVX, and was maintained at a high level, compared with the pre-operation level. The patterns of BAP and TRAP activity in the coccyges specimens were similar to those seen in the sera. The expression profiles of TRAP, RANK, and RANKL proteins in the coccyx specimens were similar to the pattern of serum TRAP activity, while the profiles of the BAP and OPG proteins were similar to the pattern of serum BAP activity in OVX rats. The changes in the mRNA expression levels of the osteogenic proteins were similar to those for protein expression. These biochemical changes in OVX rats were confirmed by immunohistochemical studies. Our results suggest that not only osteoclastogenesis accelerated but also osteoblastogenesis transiently increased during the early phase of osteoporosis.     

Brain dolichyl pyrophosphate phosphatase. Solubilization, characterization, and differentiation from dolichyl monophosphate phosphatase activity

Dolichyl [beta-32P]pyrophosphate ([beta-32P]Dol-P-P) has been prepared chemically to study Dol-P-P phosphatase in calf brain. Calf brain microsomes catalyze the enzymatic release of 32Pi from exogenous [beta-32P]Dol-P-P by a bacitracin-sensitive reaction. [32P]Pyrophosphate was not detected with the water-soluble product even when 1 mM sodium pyrophosphate was added to impede pyrophosphatase activity. A substantial fraction of the Dol-P-P phosphatase activity can be solubilized by treating brain microsomes with 3% Triton X-100. The detergent extracts catalyze the enzymatic release of 32Pi from [beta-32P]Dol-P-P and the conversion of [14C]undecaprenyl pyrophosphate to [14C]undecaprenyl monophosphate. The solubilized Dol-P-P phosphatase activity: 1) is optimal at neutral pH; 2) is inhibited by Mn2+ and stimulated by EDTA; 3) exhibits an apparent Km = 20 microM for Dol-P-P; 4) is competitively inhibited by undecaprenyl pyrophosphate, and 5) is blocked by bacitracin. Solubilized Dol-P-P phosphatase activity differs from Dol-P phosphatase activity present in the same detergent extracts with respect to: 1) thermolability at 50 degrees C, 2) effect of 20 mM EDTA, and 3) sensitivity to phosphate and fluoride ions. These studies describe the chemical synthesis of [beta-32P]Dol-P-P for use in a convenient assay of Dol-P-P phosphatase activity. A procedure for the solubilization of Dol-P-P phosphatase activity from microsomes is presented, and an enzymological comparison indicates that Dol-P-P and Dol-P phosphatase are separate enzymes in calf brain.     

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M(r) of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.     

Phosphotyrosine phosphatase: a novel phosphatase specific for phosphotyrosine, 2'-AMP and p-nitrophenylphosphate in rat brain

A unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogenous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000. The molecular weight of the native enzyme was 45,000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8 X 10(-4) M, 1.4 X 10(-4) M, and 1.7 X 10(-4) M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8 X 10(-4) M and 2.0 X 10(-4) M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L(+)tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.     

Myosin phosphatase: Structure,regulation and function

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.     

The relationship of alkaline phosphatase, CaATPase, and phytase

Alkaline phosphatase, highly purified from bovine intestinal mucosa, has significant hydrolytic activity against phytate and CaATP. Phytase and CaATPase activities require quite different assay conditions than those which are optimal for conventional alkaline phosphatase substrates such as 4-nitrophenyl phosphate. We have used affinity chromatography and antibody recognition to demonstrate that the phytase and CaATPase activities are not due to contaminating enzymes, but are intrinsic activities of intestinal alkaline phosphatase. All of the phytase and CaATPase activities present in crude extracts of bovine intestinal mucosa can be accounted for by alkaline phosphatase. Apparently neither phytase nor CaATPase exist in this tissue as independent enzymes. Specific substrates which require assay conditions quite different from the conventional 4-nitrophenyl phosphate substrate may account for the physiological function of "alkaline phosphatase."     

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.     

Radial diffusion in gel for micro determination of enzymes. I. Muramidase, alpha-amylase, DNase 1, RNase A, acid phosphatase, and alkaline phosphatase

The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference.     

Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.     

Cytochemical localization of tartrate-resistant acid phosphatase, alkaline phosphatase, and nonspecific esterase in perivascular cells of cartilage canals in the developing mouse epiphysis

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartrate-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosphatase-positive perivascular cells were present in the intermediate and deep canals adjacent to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resistant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanistic aspects of the low-molecular-weight phosphatase activity of the calmodulin-activated phosphatase, calcineurin

Product and substrate analogs have been employed as inhibitors of the low-molecular-weight phosphatase activity of calcineurin, a calmodulin-activated protein phosphatase. Product inhibition kinetics demonstrate that both products, para-nitrophenol and inorganic phosphate, inhibit para-nitrophenyl phosphate hydrolysis in a competitive manner. Inorganic phosphate is a linear competitive inhibitor, whereas the inhibition by para-nitrophenol is more complex. An analog of para-nitrophenol, pentafluorophenol, was found to be a linear competitive inhibitor. These patterns indicate a rapid equilibrium random kinetic mechanism for calcineurin. This mechanism suggests that calcineurin does not generate a phosphoryl enzyme during its catalytic reaction. Application of sulfate analogs indicates that binding of substrate occurs via the phosphoryl moiety. It is suggested that binding is a function of the affinity of ligand for the metal ion involved in calcineurin action. The dependence of the kinetic parameters of calcineurin upon pH was examined to provide information concerning the role of protonation in the activity and specificity of calcineurin. Log (VM) versus pH data for two low-molecular-weight substrates, para-nitrophenyl phosphate and tyrosine-O-phosphate, reveal a pKa value for the enzyme-substrate complex. Analysis of log (VM/KM) data yields a pKa value for the free enzyme of 8.0. Protonation of the phenolic leaving group during hydrolysis is not the rate-limiting step in calcineurin catalysis.