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At least 343 records · Page 19Linked to original sources
PubMed1982

Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis: significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver.

In experimental bile obstruction the serum activities of the membrane-bound liver enzymes, alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase are greatly increased, whereas in the liver only the alkaline phosphatase activity is elevated. After partial hepatectomy or tetrachloride poisoning the alkaline phosphatase activity in the regenerating live is increased to the same extent as in cholestasis without an accompanying elevation in serum activity. The following results support the hypothesis of a bile salt-mediated solubilization of membrane-bound enzymes in cholestatic liver: (1) 30 min after bile duct ligation the total bile acids in the liver were increased 5-fold, 2 h later as much as 10-fold. After 1 day, the bile acid concentration was still 4 times above normal. (2) Isolated plasma membranes from normal and obstructed livers were incubated in vitro with increasing amounts of tri- and dihydroxycholanic acids. At a final concentration of 1 mmol/l taurochenodeoxycholate significant amounts of membrane-bound enzymes were released into the 12,000-g supernatant. (3) In the regenerating liver, where tissue phsophatase activity was high and serum phosphatase activity unchanged, the bile salt concentration was not increased.

Alkaline Phosphatase↗
PubMed2001

Bone-specific alkaline phosphatase protein, total alkaline phosphatase activity and lactate dehydrogenase in sera of patients with sickle cell disease.

Serum bone-specific alkaline phosphatase protein (bAP) was evaluated as indicator of bone turnover by immunoradiometric assay (IRMA) in twenty patients with sickle cell disease and in twenty healthy control subjects. Serum bAP was also compared with serum total alkaline phosphatase activity and serum lactate dehydrogenase in the same group. The concentrations of serum bAP and serum lactate dehydrogenase were significantly higher in the study group than in the control group (p < 0.05, p < 0.01, respectively). The serum total alkaline phosphatase activity showed no significant difference with the control healthy subjects. There was no correlation between serum bAP and total alkaline phosphatase or lactate dehydrogenase levels in the patient group. In conclusion, serum bAP protein measured by IRMA can be considered a sensitive marker of bone turnover and could be especially useful as valuable non-invasive biochemical marker for identifying sickle cell patients with skeletal complications.

Adolescent↗
PubMed2005

Recruitment of the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase-2 to the p85 subunit of phosphatidylinositol-3 (PI-3) kinase is required for insulin-like growth factor-I-dependent PI-3 kinase activation in smooth muscle cells.

IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.

Animals↗
PubMed2003

The Drosophila dual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER.

ERK MAP kinase plays a key role in relaying extracellular signals to transcriptional regulation. As different activity levels or the different duration of ERK activity can elicit distinct responses in one and the same cell, ERK has to be under strict positive and negative control. Although numerous genes acting positively in the ERK signaling pathway have been recovered in genetic screens, mutations in genes encoding negative ERK regulators appear underrepresented. We therefore sought to genetically characterize the dual-specificity phosphatase DMKP3. First, we established a novel assay to elucidate the substrate preferences of eukaryotic phosphatases in vivo and thereby confirmed the specificity of DMKP3 as an ERK phosphatase. The Dmkp3 overexpression phenotype characterized in this assay permitted us to isolate Dmkp3 null mutations. By genetic analysis we show that DMKP3 and the tyrosine phosphatase PTP-ER perform partially redundant functions on the same substrate, ERK. DMKP3 functions autonomously in a subset of photoreceptor progenitor cells in eye imaginal discs. In addition, DMKP3 function appears to be required in surrounding non-neuronal cells for ommatidial patterning and photoreceptor differentiation.

Amino Acid Sequence↗
PubMed2002

Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein.

Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.

Amino Acid Substitution↗
PubMed1975

Phosphatases XII. Isoenzymes of alkaline phosphatase and radionuclear investigation (85Sr) of patients with neoplastic affection of the skeleton.

In a group of 30 patients with neoplastic processes, 13 were found to have a significantly increased serum alkaline phosphatase activity. This finding correlated with the results of investigation with 85Sr in 7 patients, but owing to a concomitant hepatal symptomatology, it proved of differential diagnostic value in only one of them. On the other hand, the activity of bone isoenzyme of alkaline phosphatase was significantly altered in 15 patients. In 14 of them the increase in bone isoenzyme activity corresponded to an X-ray and a radionuclear finding of a tumor process in the bones. This activity was within the normal range of values in only one patient with a positive result of the 85Sr investigation. An agreement between the results of determination of bone isoenzyme activity of alkaline phosphatase and those of radionuclear investigations was found in 27 patients. In addition, a correlation was established between the increased activity of bone isoenzyme and that of intestinal isoenzyme of alkaline phosphatase. Enzyme investigation can be suitably utilized, alongside radionuclear examination, for early detection of a bone process.

Adult↗
PubMedDate not supplied

Endogenous phosphatase activity in tumor cells in a liver aspirate. A potential problem for immunocytodiagnosis using immunoalkaline phosphatase methods.

Immunocytochemical studies were performed on fine needle aspirates of the liver in a patient with hepatocellular carcinoma. A panel of commercially available antibodies was used to study the aspirated cells by immunoalkaline phosphatase and immunoperoxidase methods. The malignant cells in the aspirates, which were positively stained by the immunoperoxidase method for alphafetoprotein and by both methods for epithelial membrane antigen, were most probably hepatocellular in origin. Some cells were shown by the immunoalkaline phosphatase method to possess leukocyte-common antigen (LCA) and antigens of colonic and ovarian tissues. These findings were further investigated, and it was found that the tumor cells indeed had LCA as well as levamisole-resistant alkaline phosphatase activity. Although the immunoalkaline phosphatase methods are useful immunodiagnostic techniques applicable to fine needle aspirates, the endogenous enzyme activity present in some nonhematopoietic tumor cells is a cause for caution in the use of these methods in aspirates from nonhematopoietic tumor tissues.

Alkaline Phosphatase↗
PubMed1977

"Prostatic acid phosphatase?" A comparison of acid phosphatase activities in epithelial cells, granulocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium.

Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells.

Acid Phosphatase↗
PubMed2005

Myotubularin phosphoinositide phosphatases, protein phosphatases, and kinases: their roles in junction dynamics and spermatogenesis.

Spermatogenesis in the seminiferous epithelium of the mammalian testis is a dynamic cellular event. It involves extensive restructuring at the Sertoli-germ cell interface, permitting germ cells to traverse the epithelium from basal to adluminal compartment. As such, Sertoli-germ cell actin-based adherens junctions (AJ), such as ectoplasmic specializations (ES), must disassemble and reassemble to facilitate this event. Recent studies have shown that AJ dynamics are regulated by intricate interactions between AJ integral membrane proteins (e.g., cadherins, alpha6beta1 integrins and nectins), phosphatases, kinases, adaptors, and the underlying cytoskeleton network. For instance, the myotubularin (MTM) phosphoinositide (PI) phosphatases, such as MTM related protein 2 (MTMR2), can form a functional complex with c-Src (a non-receptor protein tyrosine kinase). In turn, this phosphatase/kinase complex associates with beta-catenin, a constituent of the N-cadherin/beta-catenin functional unit at the AJ site. This MTMR2-c-Src-beta-catenin complex apparently regulates the phosphorylation status of beta-catenin, which determines cell adhesive function conferred by the cadherin-catenin protein complex in the seminiferous epithelium. In this review, we discuss the current status of research on selected phosphatases and kinases, and how these proteins potentially interact with adaptors at AJ in the seminiferous epithelium to regulate cell adhesion in the testis. Specific research areas that are open for further investigation are also highlighted.

Adherens Junctions↗
PubMed1988

Phosphotyrosine phosphatase activity of human and canine acid phosphatases of prostatic origin.

Human and canine prostatic specimens containing high levels of acid phosphatase (AP) activity were tested, at acid pH, for their ability to hydrolyze the major phosphoaminoacids present in phosphorylated proteins, phosphoserine (p-ser), phosphothreonine (p-thr), and phosphotyrosine (p-tyr). The cleavage of a synthetic substrate, para-nitrophenyl-phosphate (p-npp), was also measured as an indicator of AP activity; its inhibition by sodium-L-tartrate (T) was used as a criterion to identify prostatic acid phosphatase (PAP). It was found that: 1) the Km of p-tyr and p-npp were 2.0 mM and 0.41 mM, respectively, with similar Vmax values (0.078 and 0.087 mumoles of phosphate (Pi) liberated per minute per milligram of protein); 2) the ID50 were 0.25 mM and 0.50 mM with sodium orthovanadate (VO4) and T, respectively, using p-npp as substrate-with p-tyr as substrate, the values obtained were 0.016 mM and 0.11 mM, respectively; 3) activity toward p-ser and p-thr was minimal; 4) native PAP from dog seminal plasma, with a molecular weight of 90-100 kD, as determined by gel filtration on HPLC, hydrolyzed p-tyr preferentially, and this phosphatase (Pase) activity was also strongly inhibited by both T and VO4; and 5) the AP present in human and canine prostatic tissue and cells, as well as in their secretions, also preferentially hydrolyzed phosphotyrosine, and it was inhibited by T and VO4. It is proposed that these p-tyr Pases may be involved in the local regulation of prostatic growth.

Acid Phosphatase↗
PubMed1998

Studies on the protein tyrosine phosphatase activity of tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme with unknown biological function. In human tissues, its expression is restricted to bone-resorbing osteoclasts and activated macrophages. Osteoclasts secrete TRAP to the circulation during bone resorption. Reduction of the enzyme's binuclear iron center is important in regulating its activity. The purple form of the enzyme is inactive and contains two ferric ions. Mild reduction activates it to a pink form containing one ferric and one ferrous ion. Instead, strong reduction removes the iron content, resulting in a colorless, inactive enzyme. We describe spontaneous activation of the purple form to the pink form upon incubation at +37 degrees C. Further incubation results in slow inactivation of the enzyme and color change to yellowish. The enzyme purified from osteoclasts is a mixture of the purple and pink forms, but the enzyme purified from serum represents the yellowish form. We suggest that the newly synthesized enzyme is purple and reduced in the cell to the functionally active pink form. After fulfilling its biological function in the cell, the enzyme is further reduced to the yellowish form and secreted into the circulation. In the serum, further reduction would dissociate the iron content. The enzymes from osteoclasts and macrophages had similar catalytic properties, both being active as a protein tyrosine phosphatase (PTPase). The acid phosphatase (AcP) and PTPase activities were similar, and the preferred AcP substrate, pNPP, was processed in the same active site as phosphotyrosine. Our results suggest that redox-regulated PTPase activity may be a major function of TRAP in vivo.

Acid Phosphatase↗
PubMed2002

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

3T3 Cells↗
PubMed2006

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase.

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.

Amino Acid Sequence↗
PubMed1998

The major myosin phosphatase in skeletal muscle is a complex between the beta-isoform of protein phosphatase 1 and the MYPT2 gene product.

Myosin is dephosphorylated by distinct forms of protein phosphatase 1 (PP1) in smooth muscle and skeletal muscle that are composed of PP1 complexed to different regulatory subunits. The smooth muscle myosin phosphatase (smPP1M) has been characterised previously and is composed of PP1beta complexed to M110 and M21 subunits that enhance the dephosphorylation of smooth muscle myosin, but not skeletal muscle myosin. In contrast, the regulatory subunit(s) of skeletal muscle myosin phosphatase (skPP1M) greatly enhance(s) the dephosphorylation of skeletal muscle myosin. Here we identify a regulatory subunit of skPP1M as the product of the MYPT2 gene, a protein whose sequence is 61% identical to the M110 subunit of smPP1M. Surprisingly, the M21 subunit of smPP1M appears to be produced from the same gene that encodes MYPT2.

Amino Acid Sequence↗
PubMed2003

Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor.

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.

Alkaline Phosphatase↗
PubMed2003

Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats.

Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer's disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer's-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.

Animals↗
PubMed2004

FcgammaRIIb-mediated negative regulation of BCR signalling is associated with the recruitment of the MAPkinase-phosphatase, Pac-1, and the 3'-inositol phosphatase, PTEN.

The low-affinity receptor for IgG, FcgammaRIIb, negatively regulates B cell antigen receptor (BCR)-mediated proliferative signalling. FcgammaRIIb has been reported to mediate this inhibition by uncoupling the BCR from the RasMAPkinase pathway. We now show that FcgammaRIIb-mediated negative feedback inhibition also correlates with induction of an Erk-associated phosphatase activity that reflects the rapid association of Erk and the MAPkinase phosphatase, Pac-1, and dephosphorylation and inactivation of ErkMAPkinase. This mechanism of abrogating ongoing ErkMAPkinase signalling therefore provides a rationale for rapid immune-complex-mediated feedback inhibition of active antigen-driven B cell responses. In addition, FcgammaRIIb signalling also induces the recruitment and activation of the 3'-inositol phosphatase, PTEN, which by antagonising PI 3kinase activity and inhibiting BCR-coupling to the anti-apoptotic kinase, Akt, provides an additional mechanism for FcgammaRIIb-mediated negative regulation of BCR-coupling to ErkMAPkinase, cell survival and proliferation.

Animals↗
PubMed2006

Magnesium-dependent phosphatase-1 is a protein-fructosamine-6-phosphatase potentially involved in glycation repair.

Fructosamine-3-kinase (FN3K) is a recently described protein-repair enzyme responsible for the removal of fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show here that, compared with glucose, glucose 6-phosphate (Glu-6-P) reacted 3-6-fold more rapidly with proteins and 8-fold more rapidly with N-alpha-t-Boc-lysine, being therefore a more significant intracellular glycating agent than glucose in skeletal muscle and heart. Fructosamine 6-phosphates, which result from the reaction of amines with Glu-6-P, were not substrates for FN3K. However, a phosphatase that dephosphorylates protein-bound fructosamine 6-phosphates was found to be present in rat tissues. This enzyme was purified to near homogeneity from skeletal muscle and was identified as magnesium-dependent phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-bound fructosamine 6-phosphates with a catalytic efficiency >10-fold higher than those observed with its next best substrates (arabinose 5-phosphate and free fructoselysine 6-phosphate) and >100-fold higher than with protein-phosphotyrosine. It had no detectable activity on fructosamine 3-phosphates. MDP-1 dephosphorylated up to approximately 75% of the fructosamine 6-phosphates that are present on lysozyme after incubation of this protein with Glu-6-P. Furthermore, lysozyme glycated with Glu-6-P was converted by MDP-1 to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from Glu-6-P.

Amino Acid Sequence↗