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1.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,927(1):74-80
It was recently reported that the type II casein kinase of rat liver cytosol co-purified with a major 90 kDa substrate when subjected to gel filtration at low ionic strength. The identity of the 90 kDa substrate was unknown. We have verified this report and have shown that the 90 kDa substrate is recognized by a monoclonal antibody prepared against the 90 kDa heat shock protein. This ubiquitous phosphoprotein is known to increase in abundance in cells subjected to heat stress and has been shown to complex steroid receptors and certain retrovirus tyrosine kinases. 相似文献
2.
Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the α catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons. 相似文献
3.
Phosphorylation of smooth muscle caldesmon by three protein kinases: implication for domain mapping 总被引:1,自引:0,他引:1
Phosphorylation of duck gizzard caldesmon by Ca2+/phospholipid-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase and casein kinase II has been investigated. The Ca2+/phospholipid-dependent protein kinase incorporates more than 3 mol phosphate per mol (140 kDa) caldesmon. All phosphorylation sites are localized in the actin- and calmodulin-binding peptide (40-45 kDa) supposed to be a part of the C-terminal domain of caldesmon. Casein kinase II phosphorylates only one site located in a short (25-27 kDa) peptide, presumably in the caldesmon N-terminal domain. The Ca2+/calmodulin-dependent protein kinase phosphorylates two sites located in the N- and C-terminal domains of caldesmon. 相似文献
4.
Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues. 总被引:2,自引:0,他引:2
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We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species. 相似文献
5.
Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species. 相似文献
6.
Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.
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Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase. 相似文献
7.
Brendan W. Wren Peter P. Mullany Christopher L. Clayton Soad Tabaqchali 《FEMS microbiology letters》1988,47(3):163-168
Abstract Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile . A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated λCd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone λCd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against λCd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species. 相似文献
8.
In the course of searching for proteins that interact with protein kinase B in 3T3-L1 adipocytes, we isolated a 105 kDa protein from 3T3-L1 adipocytes. Peptides sequenced from the protein were found to be present in several expressed sequence tags. A cDNA containing one of these expressed sequence tags was sequenced and appears to contain the entire coding region. Computer analysis revealed a potential protein kinase domain at the N-terminus; however, the first subdomain and several invariant residues characteristic of protein kinases are absent. An antibody was raised against a peptide from the 105 kDa protein. By immunoblotting, it was found that the protein was widely expressed in mouse tissues, and concentrated in the cytosol and low density microsome fractions of 3T3-L1 adipocytes. 相似文献
9.
Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species. 相似文献
10.
Anti-peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex 总被引:1,自引:1,他引:0
Gerard Peaucellier Kirsten Shartzer Wanping Jiang Katia Maggio William H. Kinsey 《Development, growth & differentiation》1993,35(2):199-208
The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose-coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane. 相似文献
11.
Steven Pelech Harry Paddon Linda Kwong Gerald Weeks 《Development, growth & differentiation》1989,31(4):351-361
Cell-free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate-associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S-300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE-Sephacel and heparin-Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE-Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa. 相似文献
12.
A type-1 casein kinase from yeast phosphorylates both serine and threonine residues of casein. Identification of the phosphorylation sites 总被引:1,自引:0,他引:1
A Donella-Deana N Grankowski W Kudlicki R Szyszka E Gasior L A Pinna 《Biochimica et biophysica acta》1985,829(2):180-187
A protein kinase (casein kinase 1A) active on casein and phosvitin but not on histones has been purified to near homogeneity from yeast cytosol and meets most criteria for being considered a type-1 casein kinase: it is a monomeric enzyme exhibiting an Mr of about 27 kDa by sucrose gradient centrifugation: it is not affected by inhibitors of type-2 casein kinases, such as heparin and polyglutamate, and shows negligible affinity for GTP. It also readily phosphorylates the residue Ser-22 of beta-casein located within the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser22-Ile-Thr-Arg- which is typically affected by casein kinases of the first class. On the other hand, casein kinase 1A displays the unusual property of phosphorylating threonine residue(s) in both whole casein and alpha s1-casein. The threonine residue phosphorylated in alpha s1-casein and accounting for most of the 32P incorporated into this protein by casein kinase 1A has been identified as Thr-49, which occurs in the sequence -Ser(P)-Glu-Ser(P)-Thr(P*)49-Glu-Asp-Gln-, whose two Ser(P) residues are already phosphorylated in the native protein. It is concluded that some type-1 casein kinases can also phosphorylate threonine residues provided they fulfil definite structural requirements, probably an acidic cluster near their N-terminal side. 相似文献
13.
K V Kandror A O Vasil'ev D V Kapkov A M Fuéntes A S Stepanov 《Biokhimii?a (Moscow, Russia)》1990,55(12):2115-2121
Homogeneous preparations of casein kinases 2 have been isolated from bovine liver and the phytopathogenic fungus Verticillium dahliae. The isolation procedure was similar in both cases and included consecutive chromatography on heparin-Sepharose, phosphocellulose PII and monoQ. The bovine liver enzyme consists of two subunits with molecular masses of 38 kDa and 27 kDa, while the fungus kinase has three subunits with Mr of 53 kDa, 41 kDa and 38 kDa. Self-phosphorylation of casein kinase 2 in vitro resulted in phosphate incorporation into the smaller subunit of the mammalian enzyme and into all the three subunits of the fungal enzyme. The Km value of casein kinase 2 from bovine liver for ATP is 3.1 microM; that for GTP is 22.0 microM. The corresponding parameters of the fungal kinase are 14.3 and 18.2 microM, respectively. Specific antibodies to each kinase have been raised. The lack of antigenic cross-reactions between the two enzymes has been demonstrated by ELISA. 相似文献
14.
Zona pellucida (ZP) binding proteins from boar spermatozoa were compared with antigens recognized by ACR.2 and ACR.3 monoclonal antibodies. The ZP binding proteins of 55, 53, 45 and 38 kDa are identical with various forms of boar acrosin immunologically detected by ACR.2 antibody. The ZP binding proteins of 17 and 18 kDa are recognized by ACR.3 antibody. The N-terminal amino acid sequence of the 17 kDa protein revealed that it is not derived from an acrosin molecule. 相似文献
15.
Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes. 总被引:1,自引:0,他引:1
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The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species. 相似文献
16.
Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells 总被引:1,自引:0,他引:1
We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I. 相似文献
17.
Katsumi Shinkawa Shigeo Nakajo Kazuyasu Nakaya Yasuharu Nakamura 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,930(3)
We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I. 相似文献
18.
The vacuolar H(+)-ATPase (V-ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells. Although a 100 kDa subunit is common to many V-ATPases, it is not detected in a purified and active pump from oat (Ward J.M. and Sze H. (1992) Plant Physiol. 99, 925-931). A 100 kDa subunit of the yeast V-ATPase is encoded by VPH1. Immunostaining revealed a Vph1p-related polypeptide in oat membranes, thus the role of this polypeptide was investigated. Membrane proteins were detergent-solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100 kDa polypeptide was not associated with the fully assembled ATPase; however, it was part of an approximately 250 kDa V0 complex including subunits of 36 and 16 kDa. Immunostaining with an affinity-purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the approximately 670 kDa holoenzyme. Co-immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol. The free V1 subcomplex became attached to the detergent-solubilized V0 sector after mixing, as subunits of both sectors were co-precipitated by an antibody against subunit A. The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity. Its association with the free Vo subcomplex would support a role of this protein in V-ATPase assembly and perhaps in sorting. 相似文献
19.
Merozoites were isolated from Plasmodium falciparum cultures labeled with [3H]mannose and [35S]methionine and treated with a cleavable homobifunctional crosslinker, dithiobis(succinimidyl) propionate. The crosslinked complexes were immunoprecipitated with Mab.5B1 directed against the major merozoite surface glycoprotein. Pf200 (MW 190-205), and reduced with dithiothreitol. Crosslinked immunocomplexes did not contain the second major merozoite surface glycoprotein, Pf50 (MW 45-55 kDa), or other major [35S]methionine-labeled proteins, except for a weakly labeled protein of 150 kDa. Crosslinked complexes immunoprecipitated with Mab.5B1 and then reduced with DTT were immunoblotted with antibody directed against three soluble P. falciparum antigens, a serine-rich antigen known as Pf126 or SERA, the S-antigen, and GBP-130. The 150-kDa S-antigen was readily detected in crosslinked immunocomplexes with Pf200. The SERA antigen, although crosslinked under these conditions, was not detected in association with Pf200 nor was GBP-130. 相似文献
20.
Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins 总被引:2,自引:0,他引:2
Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins. 相似文献