Phosphoprotein phosphatase of human central nervous system myelin: purification to apparent homogeneity of a low Mr phosphatase and characterization of the high Mr phosphatase.
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Brain↗
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Specificity of a rat liver protein phosphatase active on caseins phosphorylated by two cAMP-independent protein kinases. "Casein phosphatase" and the 'multifunctional' phosphoprotein phosphatase are different enzymes.
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Animals↗
Studies on the plasma phosphatase of normal and rachitic chicks; relationship between plasma phosphatase and the phosphatases of bone, kidney, liver and intestinal mucosa.
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Animals↗
Studies on human prostatic acid phosphatase. III. The effects of alcohols on human erythrocyte acid phosphatase and on human prostatic acid phosphatase.
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Acid Phosphatase↗
Phosphatases, X. Study on the mechanism of induction of alkaline phosphatase activity in rat liver after bile flow obstruction. Purification of the inducible liver alkaline phosphatase and incorporation of radioactive precursors.
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Alkaline Phosphatase↗
[Studies on experimental stimulation of ossification in the paradental tissues of rats and blood level of Ca, P and alkaline phosphatase in them. 1. Histological reactions of paradental tissues to administration of dehydrotachysterol. 2. Effect of dehydrotachysterol on blood level of Ca, P and alkaline phosphatase. 3. Effect of dehydrotachysterol on blood level of Ca, P and alkaline-phosphatase in rats subjected to abnormal sexual conditions].
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Alkaline Phosphatase↗
Multiple forms of phosphatase from human brain: isolation and partial characterization of affi-gel blue nonbinding phosphatase activities.
Phosphatases extracted from a human brain were resolved into two main groups, namely affi-gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue binding phosphatases were further separated into four different phosphatase activities, designated P1-P4, and described previously. In the present study we describe the affi-gel blue-nonbinding phosphatases which were separated into seven different phosphatase activities, designated P5-P11 by poly-(L-lysine)-agarose and aminohexyl Sepharose 4B chromatographies. These seven phosphatase activities were active toward nonprotein phosphoester. P7-P11 and to some extent P5 could also dephosphorylate a phosphoprotein. They displayed different enzyme kinetics. On the basis of activity peak, the apparent molecular mass as estimated by Sephadex G-200 column chromatography for P5 was 49 kDa; P6, 32 kDa; P7, 150 kDa; P8, 250 kDa; P9, 165 kDa; P10, 90 kDa and P11, 165 kDa. Immunoblot analysis indicated that P8-P11 may belong to PP2B family, whereas P7 may associate with PP2A. The phosphatases P7-P11 were found to be effective in the dephosphorylation of Alzheimer's disease abnormally hyperphosphorylated tau. The resulting dephosphorylated tau regained its activity in promoting the microtubule assembly, suggesting that P7-P11 might regulate the phosphorylation of tau protein in the brain.
Aged↗
3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase.
The presence of a nonspecific acid phosphatase which had high activity with 3-phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3-phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2-phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation.
Journal Article↗
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)
Acid Phosphatase↗
Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity.
The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.
Acid Phosphatase↗
Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.
We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.
3T3 Cells↗
Control of the mutagenicity of aromatic amines by protein kinases and phosphatases. I. The protein phosphatase inhibitors okadaic acid and ortho-vanadate drastically reduce the mutagenicity of aromatic amines.
The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10(-9)-10(-6) M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by > 80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of 32P-labelled phosphate from intracellularly prelabelled [32P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.
2-Acetylaminofluorene↗
Differential modulation of cortical synaptic activity by calcineurin (phosphatase 2B) versus phosphatases 1 and 2A.
Reversible protein phosphorylation is thought to play an important regulatory role in synaptic neurotransmission. We recently have shown in cultured rat cortical neurons that inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin (phosphatase 2B) increases the frequency, but not the amplitude, of postsynaptic glutamatergic currents, implicating a presynaptic site of action for calcineurin. The specific presynaptic phosphoprotein substrates for calcineurin are unknown, however, calcineurin has been implicated in the control of the Ca2+-independent phosphatases, phosphatases 1 and 2A. To determine whether calcineurin's effects on synaptic transmission are direct or are mediated by changes in phosphatase 1 and/or 2A activities, we used whole-cell voltage clamp to record spontaneous and miniature excitatory postsynaptic currents in the presence of calyculin A (1 microM in bath solution), a membrane permeant inhibitor of phosphatases 1 and 2A which has no effect on calcineurin. Calyculin increased postsynaptic current amplitude without changing current frequency. In these same neurons, subsequent inhibition of calcineurin with cyclosporine A or FK506 had no further effect on current amplitude, but increased current frequency. The increased current amplitude seen with calyculin involved a postsynaptic mechanism, since the effect was reproduced by microcystin (10 microM in pipette solution), which is a membrane-impermeant inhibitor of phosphatases 1 and 2A. Thus, in rat cortical neurons, glutamatergic neurotransmission appears to be frequency-modulated through a presynaptic mechanism by calcineurin, and amplitude-modulated through a postsynaptic mechanism by phosphatases 1 and 2A.
Amino Acid Sequence↗
Identification of protein phosphatase 1 as a mitotic lamin phosphatase.
At the onset of mitosis, the nuclear lamins are hyperphosphorylated leading to nuclear lamina disassembly, a process required for nuclear envelope breakdown and entry into mitosis. Multiple lamin kinases have been identified, including protein kinase C, that mediate mitotic lamin phosphorylation and mitotic nuclear lamina disassembly. Conversely, lamin dephosphorylation is required for nuclear lamina reassembly at the completion of mitosis. However, the protein phosphatase(s) responsible for the removal of mitotic phosphates from the lamins is unknown. In this study, we use human lamin B phosphorylated at mitosis-specific sites as a substrate to identify and characterize a lamin phosphatase activity from mitotic human cells. Several lines of evidence demonstrate that the mitotic lamin phosphatase corresponds to type 1 protein phosphatase (PP1). First, mitotic lamin phosphatase activity is inhibited by high nanomolar concentrations of okadaic acid and the specific PP1 peptide inhibitor, inhibitor-2. Second, mitotic lamin phosphatase activity cofractionates with PP1 after ion exchange chromatography. Third, microcystin-agarose depletes mitotic extracts of both PP1 and lamin phosphatase activity. Our results demonstrate that PP1 is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates from lamin B, a process required for nuclear lamina reassembly.
Chromatography, Ion Exchange↗
The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle.
The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
Adenosine Triphosphate↗
The phospho-opsin phosphatase from bovine rod outer segments. An insight into the mechanism of stimulation of type-2A protein phosphatase activity by protamine.
The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of alpha subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 micrograms/ml; 50 microM). However, when phospho-peptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCl, whence the protamine-dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.
Amino Acid Sequence↗
Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle.
Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.
Animals↗
Have we overlooked the importance of serine/threonine protein phosphatases in pancreatic beta-cells? Role played by protein phosphatase 2A in insulin secretion.
Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.
Calcineurin↗