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1.
CD45-protein tyrosine phosphatase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells 总被引:10,自引:0,他引:10
Activation of peripheral blood T cells, and the leukemic T cell line Jurkat, as measured by mobilization of intracellular calcium, by an anti-TCR antibody is blocked by mAb (T191) to the leukocyte common Ag (CD45). T191 also blocked down-regulation of the CD3-TCR complex induced by an anti-CD3 mAb. Vanadate, a phosphotyrosine phosphatase inhibitor, partially blocks the effect of T191 and restored mobilization of intracellular calcium. Assays of the immunoprecipitates of T191 and CD45 from both Jurkat-BM1 and peripheral T cells showed that the immune complexes had intrinsic phosphatase activity. A parallel immunoprecipitate using a mAb (4-10) against HLA class I showed no such activity. Further analysis of the T191 immunocomplex revealed activity against phosphotyrosine, p-nitrophenylphosphate, and [32P-poly-glu-tyr, but not against phosphoserine. Phosphatase activity was inhibited by Vanadate, but not by Zn2+ or F-. These results show that CD45 is a phosphotyrosine phosphatase, and strongly suggest that tyrosine phosphorylation/dephosphorylation is critically involved in activation of T cells through the TCR-CD3 complex. 相似文献
2.
A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions. 相似文献
3.
J H Kim H J Do W H Wang Z Macháty Y M Han B N Day R S Prather 《Biology of reproduction》1999,61(4):900-905
This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization. 相似文献
4.
P W Pappas 《Experimental parasitology》1991,72(4):362-367
The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate (PPi), and beta-glycerophosphate (beta GP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants (Ki')): molybdate (0.031 mM); PPi (0.147 mM); NaF (0.150 mM); o-carboxyphenyl phosphate (0.261 mM); inorganic phosphate (0.770)); arsenate (3.45 mM); tartrate (22.1 mM); and beta GP (29.8 mM). Cu2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis (Vmax) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant (Km') for PNPP increased from 0.233 to 0.351 mM when the pH was raised from 4.0 to 5.0. The Km' for PPi decreased from 0.101 to 0.046 mM, while the Km' for beta GP changed from 2.04 to 2.22 mM under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 degrees C. 相似文献
5.
Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity 总被引:1,自引:0,他引:1
The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid. 相似文献
6.
Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity. 相似文献
7.
E Paramithiotis L Tkalec M J Ratcliffe 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(11):3710-3717
In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation. 相似文献
8.
Hydrolysis of the tail phosphotyrosine in Src family members is catalyzed by the protein-tyrosine phosphatase CD45, activating Src family-related signaling pathways. Using purified recombinant phospho-Src (P-Src) (amino acid residues 83-533) and purified recombinant CD45 catalytic (cytoplasmic) domain (amino acid residues 565-1268), we have analyzed the kinetic behavior of dephosphorylation. A time course of phosphatase activity showed the presence of a burst phase. By varying the concentration of P-Src, it was shown that the amplitude of this burst phase increased linearly with respect to P-Src concentration. Approximately 2% of P-Src was shown to be rapidly dephosphorylated followed by a slower linear phase. A P-Src protein substrate containing a functional point mutation in the Src homology domain 2 (SH2) led to more rapid dephosphorylation catalyzed by CD45, and this reaction showed only a single linear kinetic phase. These results were interpreted in terms of a model in which P-Src exists in a relatively slow dynamic equilibrium between "closed" and "open" conformational forms. Combined mutations in the SH2 and SH3 domain or the addition of an SH3 domain ligand peptide enhanced the accessibility of P-Src to CD45 by biasing P-Src to a more open form. Consistent with this model, a phosphotyrosine peptide that behaved as an SH2 domain binding ligand showed approximately 100-fold greater affinity for unphosphorylated Src versus P-Src. Surprisingly, P-Src possessing combined SH3 and SH2 functional inactivating point mutations was dephosphorylated by CD45 more slowly compared with P-Src completely lacking SH3 and SH2 domains. Additional data suggest that the SH3 and SH2 domains can inhibit accessibility of the P-Src tail to CD45 by interactions other than direct phosphotyrosine binding by the SH2 domain. Taken together, these results suggest how activation of Src family member signaling pathways by CD45 may be influenced by the presence or absence of ligand interactions remote from the tail. 相似文献
9.
We have examined the interaction of hepatic phenylalanine hydroxylase with the phenylalanine analogs, tryptophan and the diastereomers of 3-phenylserine (beta-hydroxyphenylalanine). Both isomers of phenylserine are substrates for native phenylalanine hydroxylase at pH 6.8 and 25 degrees C, when activity is measured with the use of the dihydropteridine reductase assay coupled with NADH in the presence of the synthetic cofactor, 6-methyl-5,6,7,8-tetrahydropterin. However, while erythro-phenylserine exhibits simple Michaelis-Menten kinetics (Km = 1.2 mM, Vmax = 1.2 mumol/min X min) under these conditions, the threo isomer exhibits strong positive cooperativity (S0.5 = 4.8 mM Vmax = 1.4 mumol/min X mg, nH = 3). Tryptophan also exhibits cooperativity under these conditions (S0.5 = 5 mM, Vmax = 1 mumol/min X mg, nH = 3). The presence of 1 mM lysolecithin results in a hyperbolic response of phenylalanine hydroxylase to tryptophan (Km = 4 mM, Vmax = 1 mumol/min X mg) and threo-phenylserine (Km = 2 mM, Vmax = 1.4 mumol/min X mg). erythro-Phenylserine is a substrate for native phenylalanine hydroxylase in the presence of the natural cofactor, L-erythro-tetrahydrobiopterin (BH4) (Km = 2 mM, Vmax 0.05 mumol/min X mg, nH = 2). Preincubation of phenylalanine hydroxylase with erythro-phenylserine results in a 26-fold increase in activity upon subsequent assay with BH4 and erythro-phenylserine, and hyperbolic kinetic plots are observed. In contrast, both threo-phenylserine and tryptophan exhibit negligible activity in the presence of BH4 unless the enzyme has been activated. The product of the reaction of phenylalanine hydroxylase with either isomer of phenylserine was identified as the corresponding p-hydroxyphenylserine by reaction with sodium periodate and nitrosonaphthol. With erythro-phenylserine, the hydroxylation reaction is tightly coupled (i.e. 1 mol of hydroxyphenylserine is formed for every mole of tetrahydropterin cofactor consumed), while with threo-phenylserine and tryptophan the reaction is largely uncoupled (i.e. more cofactor consumed than product formed). Erythro-phenylserine is a good activator, when preincubated with phenylalanine hydroxylase (A0.5 = 0.2 mM), with a potency about one-third that of phenylalanine (A0.5 = 0.06 mM), while threo-phenylserine (A0.5 = 6 mM) and tryptophan (A0.5 approximately 10 mM) are very poor activators. Addition of 4 mM tryptophan or threo-phenylserine or 0.2 mM erythro-phenylserine to assay mixtures containing BH4 and phenylalanine results in a dramatic increase in the hydroxylation at low concentrations of phenylalanine.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
10.
About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells. 相似文献
11.
K D Hammond R K Wollbrandt D A Gilbert 《The International journal of biochemistry》1985,17(2):259-264
The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM). 相似文献
12.
S J Goldman S Uniyal L M Ferguson D E Golan S J Burakoff P A Kiener 《The Journal of biological chemistry》1992,267(9):6197-6204
The effect of two anti-CD45 (T200, LCA, Ly5) antibodies on the activation of the murine T-cell hybridoma 13.13 has been evaluated. These studies have been carried out in a system that did not require cross-linking or coclustering of antibodies. Activation of 13.13 cells with the anti-CD3 monoclonal antibody, 145.2C11, gave rise to rapid increases in intracellular calcium and interleukin-2 production. Additionally, within 1 min, phosphorylation on tyrosine of four major proteins of about 130,000, 110,000, 80,000, and 37,000 daltons could be seen. Pretreatment of the cells with the anti-CD45 mAb M1/89.18.7.HK markedly inhibited all three biological responses, while an alternate anti-CD45 antibody, M1/9.3.4.HL.2, had little effect. The two antibodies bound to CD45 with similar affinities, and no differences in the lateral mobility of antibody-CD45 complexes in the cell membrane were observed. The inhibition of activation of the cells by M1/89.18.7.HK was abrogated significantly both by the phosphotyrosine protein phosphatase inhibitor orthovanadate and by excess M1/9.3.4.HL.2. If M1/89.18.7.HK was added to the 13.13 cells after they had already been activated with anti-CD3, it very effectively stimulated dephosphorylation of substrates that had been phosphorylated on tyrosines prior to adding the anti-CD45 antibody. These results indicate that the phosphotyrosine protein phosphatase activity of CD45 is critical to its biological function and that bivalent (i.e. uncross-linked) anti-CD45 antibodies can give rise to markedly different responses. One of the antibodies, M1/89.18.7.HK, appears to behave much like a receptor ligand and is able to activate the enzymatic activity associated with the CD45 transmembrane protein. 相似文献
13.
de Almeida-Amaral EE Belmont-Firpo R Vannier-Santos MA Meyer-Fernandes JR 《Experimental parasitology》2006,114(4):334-340
We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity. 相似文献
14.
Meyer-Fernandes JR da Silva-Neto MA Soares Mdos S Fernandes E Vercesi AE de Oliveira MM 《Zeitschrift für Naturforschung. C, Journal of biosciences》1999,54(11):977-984
Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-amino acids and phosphoproteins under physiological conditions. 相似文献
15.
Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase 总被引:4,自引:0,他引:4
C P Chan B Gallis D K Blumenthal C J Pallen J H Wang E G Krebs 《The Journal of biological chemistry》1986,261(21):9890-9895
Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates. 相似文献
16.
Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains 总被引:10,自引:1,他引:9
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《The Journal of cell biology》1996,135(6):1515-1523
p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated. 相似文献
17.
Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma 总被引:2,自引:0,他引:2
The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis. 相似文献
18.
In experiments with (CBA x C57B1/6)F1 mice it was shown that LDH activity moderately increased 5 min after exposure of the head to 200 Gy gamma radiation. After 60 min, there was a 24.4 per cent decrease in alkaline phosphatase activity and a 24.3 per cent increase in SDG activity. Injected prior to irradiation meksamine precluded the postirradiation increase in SDH and alleviated the postirradiation decrease in alkaline phosphatase. 相似文献
19.
Florio T Arena S Thellung S Iuliano R Corsaro A Massa A Pattarozzi A Bajetto A Trapasso F Fusco A Schettini G 《Molecular endocrinology (Baltimore, Md.)》2001,15(10):1838-1852
The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP eta whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP eta in somatostatin's effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP eta) recovered somatostatin's ability to inhibit cell proliferation, showing dose-dependence and time course similar to those observed in PC Cl3 cells. Conversely, the transfection of a catalytically inactive mutant of r-PTP eta did not restore the antiproliferative effects of somatostatin. PC mos/PTP eta cells showed a high basal phosphotyrosine phosphatase activity which, similarly to PC Cl3 cells, was further increased after somatostatin treatment. The specificity of the role of r-PTP eta in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP eta activity in PCCl3 and PC mos/PTP eta (+300%, P < 0.01) but not in PCmos cells. Conversely, no differences in somatostatin-stimulated SHP-2 activity, (approximately +50%, P < 0.05), were observed among all the cell lines. The activation of r-PTP eta by somatostatin caused, acting downstream of MAPK kinase, an inhibition of insulin-induced ERK1/2 activation with the subsequent blockade of the phosphorylation, ubiquitination, and proteasome degradation of the cyclin-dependent kinase inhibitor p27(kip1). Ultimately, high levels of p27(kip1) lead to cell proliferation arrest. In conclusion, somatostatin inhibition of PC Cl3 cell proliferation requires the activation of r-PTP eta which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27(kip1). 相似文献
20.
We developed a sensitive fluorometric assay to study in vitro fusion between early endosomes isolated from the human hepatoma, Hep G2. Biochemical characterization of this assay showed that fusion between endosomal vesicles was dependent on physiologic temperature, cytosol, and ATP. Fusion was inhibited by pretreatment of vesicles and cytosol with either 1 mM N-ethylmaleimide or 20 microM GTP gamma S. Neither 3 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid nor 1 mM CaCl2 significantly affected fusion. In addition, ATP gamma S neither inhibited fusion at 50 microM nor supported fusion at 5 mM. To further our understanding of the factors regulating fusion, inhibitors of endoprotease activity and phosphotyrosine phosphatase activity were assayed for their effect on fusion. The dipeptide inhibitor of endoprotease activity, Cbz-gly-phe-amide, inhibited fusion 70% at 3 mM whereas a dipeptide analogue, Cbz-gly-gly-amide, was without effect. Furthermore, orthovanadate, an inhibitor of phosphotyrosine phosphatase activity, stimulated fusion twofold at 0.5 mM. These results suggest that both tyrosine dephosphorylation and endoprotease activity contribute to the regulation of endosome fusion. 相似文献