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1.
Proc Natl Acad Sci U S A ; 96(22): 12536-41, 1999 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-10535957

RESUMO

Human cytomegalovirus (CMV), a herpesvirus that causes congenital disease and opportunistic infections in immunocompromised individuals, encodes functions that facilitate efficient viral propagation by altering host cell behavior. Here we show that CMV blocks apoptosis mediated by death receptors and encodes a mitochondria-localized inhibitor of apoptosis, denoted vMIA, capable of suppressing apoptosis induced by diverse stimuli. vMIA, a product of the viral UL37 gene, inhibits Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release, while residing in mitochondria and associating with adenine nucleotide translocator. These functional properties resemble those ascribed to Bcl-2; however, the absence of sequence similarity to Bcl-2 or any other known cell death suppressors suggests that vMIA defines a previously undescribed class of anti-apoptotic proteins.


Assuntos
Apoptose/genética , Citomegalovirus/genética , Proteínas Estruturais Virais/genética , Linhagem Celular , Citomegalovirus/fisiologia , Células HeLa , Humanos , Replicação Viral/genética
2.
J Biol Chem ; 273(28): 17926-32, 1998 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-9651399

RESUMO

The BCL-2 proto-oncogene contains unusually long untranslated 5' and 3' sequences. Deletion of the sequences flanking the BCL-2 open reading frame dramatically increases the level of protein expression. Transient high level BCL-2 protein expression mediated by plasmid transfection or by infection with recombinant adenovirus results in potent apoptosis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5'- and 3'-flanking sequences contribute to the negative modulation of protein expression from the BCL-2 open reading frame. It appears that these sequences exert the negative regulatory effect in an orientation-dependent manner. Analysis of BCL-2 RNA levels indicate that elevated levels of mRNA may be the primary cause of elevated levels of protein expression. Apoptosis induced by adenovirus vectors expressing elevated levels of BCL-2 can be readily inhibited by the caspase inhibitor z-VAD-fmk, suggesting that high levels of BCL-2 expression induce apoptosis via the caspase cascade. Mutational analysis of BCL-2 indicates that its pro-apoptotic activity is separable from its anti-apoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in the pro-apoptotic activity, in addition to other activating mutations that result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.


Assuntos
Apoptose/genética , Caspases , Genes bcl-2 , Adenoviridae/genética , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Proteínas de Caenorhabditis elegans , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Primers do DNA , Vetores Genéticos , Humanos , Mutagênese , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética
3.
Oncogene ; 13(4): 739-48, 1996 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-8761295

RESUMO

A fusion gene consisting of wild-type p53 linked to a modified ligand binding domain of the murine estrogen receptor has been constructed and should be a useful tool for studying controlled activation of wild-type p53 function in a variety of experimental cell systems. The protein product of this gene, p53ERTM, is expressed in cells constitutively but is not functional unless associated with tamoxifen or 4-hydroxytamoxifen. p53ERTM was introduced into p53-deficient mouse embryo fibroblasts (MEFs) expressing the E1A and T24 H-ras oncogenes. Activation of p53 in these transformed cells by the addition of tamoxifen or 4-hydroxytamoxifen resulted in apoptosis. In addition to engaging the apoptotic machinery, the tamoxifen-activated fusion protein exhibited other functions characteristic of wild-type p53, such as induction of WAF1 and MDM2 gene expression and activation of the p53-dependent spindle checkpoint in cells treated with nocodazole. Activation of p53ERTM expressed in p53-positive MEFs coexpressing E1A and ras had, at most, only a small cytotoxic effect. When three cell lines of transformed p53+/+ fibroblasts not expressing p53ERTM were tested for sensitivity to the DNA-damaging drug doxorubicin, the p53+/+ clones displayed either comparable sensitivity, or at most an increase in drug sensitivity of less than fourfold, as compared to several p53-/- cell lines. Our data show that restoration of wild-type p53 activity is sufficient to trigger apoptosis in p53-/- MEFs transformed with E1A and T24 H-ras and suggest that rare propagable clones of p53-normal MEFs expressing the E1A and T24 H-ras oncogenes have suffered compensatory alterations that compromise the ability to undergo p53-dependent apoptosis.


Assuntos
Proteínas E1A de Adenovirus/genética , Apoptose/genética , Genes ras , Proteínas Recombinantes de Fusão/genética , Tamoxifeno/análogos & derivados , Animais , Linhagem Celular Transformada , Embrião de Mamíferos/citologia , Antagonistas de Estrogênios/farmacologia , Fibroblastos , Regulação da Expressão Gênica , Camundongos , Receptores de Estrogênio/genética , Tamoxifeno/farmacologia , Proteína Supressora de Tumor p53/genética
4.
Oncogene ; 12(6): 1181-7, 1996 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-8649819

RESUMO

To search for candidate genes involved in p53-mediated apoptosis, the differential display technique was used to identify RNA species whose expression was altered in murine NIH3T3 cells treated with the cytotoxic drug etoposide. We report here the isolation and characterization of EI24, a novel gene whose 2.4 kb mRNA is induced following etoposide treatment. Induction of EI24 mRNA by etoposide required expression of wild-type p53 in murine embryonic fibroblasts which had been transformed with the oncogenes E1A and T24 H-ras; and overexpression of functional p53 in these cells was sufficient to induce expression of the EI24 mRNA. The EI24 mRNA was also induced in a p53-dependent manner by ionizing irradiation of primary murine thymocytes. Isolation of a full-length EI24 cDNA revealed that its protein product bears homology to CELF37C12.2, a Caenorhabditis elegans protein of unknown function.


Assuntos
Dano ao DNA , Etoposídeo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes p53 , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Células 3T3/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genes ras , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Transformação Genética
5.
J Biol Chem ; 270(21): 12933-40, 1995 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-7759553

RESUMO

Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between 125I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.


Assuntos
Fosfoproteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo , Sequência de Aminoácidos , Compartimento Celular , Nucléolo Celular/química , Reagentes de Ligações Cruzadas , Retículo Endoplasmático/química , Imunofluorescência , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Ligação Proteica , Proteína Ribossômica L10 , Proteínas Ribossômicas/química , Ribossomos/química , Análise de Sequência , Células Tumorais Cultivadas
6.
Anal Biochem ; 224(1): 39-50, 1995 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-7535987

RESUMO

A procedure has been developed for measuring antibody binding to cell surface antigens using an immobilized plasma membrane fraction. In this method, isolated plasma membranes are dried onto wells of a 96-well microtiter plate and incubated with antibodies that recognize a cell surface protein. Bound antibody is detected indirectly using an enzyme-linked or fluorescently tagged second antibody. Alternatively, the primary antibody itself can be labeled and its binding can be detected directly. The assay is simple and fast and provides several advantages over whole cell binding assays currently in widespread use.


Assuntos
Anticorpos/metabolismo , Antígenos de Superfície/metabolismo , Animais , Anticorpos/análise , Anticorpos Monoclonais/imunologia , Antígenos CD/metabolismo , Antígenos CD19 , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Ligação Competitiva , Antígeno CD56 , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos
7.
Ciba Found Symp ; 176: 198-211; discussion 211-4, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8299420

RESUMO

VPS1 encodes a 79 kDa protein required for the proper sorting of soluble vacuolar proteins in Saccharomyces cerevisiae. The N-terminal half of Vps1p, which contains a consensus GTP-binding motif, shares extensive homology with a growing family of high molecular mass GTP-binding proteins. Members of this family have been implicated in a number of cellular processes. Vps1p most closely resembles the microtubule-associated protein dynamin. As predicted from the sequence, Vps1p binds and hydrolyses GTP. However, no requirement for microtubules was found for Vps1p function in protein sorting. In subcellular fractionation experiments Vps1p associates with the membrane fraction; the C-terminal half of Vps1p is important for this association. Mutational analysis of VPS1 generated two classes of mutations, dominant negative and recessive. The dominant mutations all mapped to the N-terminal half of the protein. Recessive mutations gave rise to either truncated or unstable proteins. A potential Vps1p-interacting protein (Mvp1p) has been isolated by screening for suppressors of the dominant alleles of VPS1. Taken together these results suggest that Vps1p is a two-domain protein that is part of a multi-subunit protein complex involved in vacuolar protein sorting.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/fisiologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Dinaminas , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Transporte Vesicular
8.
Mol Biol Cell ; 3(12): 1389-402, 1992 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1493335

RESUMO

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.


Assuntos
Proteínas Fúngicas/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Carboxipeptidases/metabolismo , Catepsina A , Teste de Complementação Genética , Genótipo , Processamento de Proteína Pós-Traducional , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae , Vacúolos/ultraestrutura
9.
J Cell Biol ; 119(4): 773-86, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1429836

RESUMO

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , ATPase de Ca(2+) e Mg(2+)/química , Proteínas de Transporte/química , Proteínas de Transporte/genética , Dinaminas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genes Fúngicos , Guanosina Trifosfato/metabolismo , Microtúbulos/fisiologia , Mutagênese , Fenótipo , Mutação Puntual , Saccharomyces cerevisiae/genética , Temperatura , Proteínas de Transporte Vesicular
10.
Mol Reprod Dev ; 25(3): 215-26, 1990 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1691919

RESUMO

Monoclonal antibodies were raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus. One of the monoclonal antibodies (MAb 69-10, an IgA) was shown by immunofluorescence labeling of intact and detergent-lysed CVs to be directed against a CV content antigen. Immunoblot analysis of CVs revealed that MAb 69-10 bound to a major CV polypeptide with an Mr similar to that of hyalin (i.e., 300,000). MAb 69-10 was subsequently shown to bind to purified hyalin prepared from S. purpuratus and to cross react with hyalin prepared from Lytechinus pictus. Immunogold labeling on thin sections of unfertilized S. purpuratus eggs showed that hyalin was localized to the electron-lucent portion of CVs. This result is in agreement with the labeling pattern obtained by Hylander and Summers (Dev Biol 93:368-380, 1982) using polyclonal antihyalin antibodies. In fertilized eggs and later-stage embryos, hyalin was observed to be located on the external surface of the embryo. MAb 69-10 should be useful in studies of the structure of hyalin and its function in morphogenesis.


Assuntos
Embrião não Mamífero/metabolismo , Hialina/metabolismo , Óvulo/metabolismo , Animais , Anticorpos Monoclonais , Antígenos , Reações Cruzadas , Grânulos Citoplasmáticos/metabolismo , Embrião não Mamífero/ultraestrutura , Feminino , Hialina/imunologia , Imuno-Histoquímica , Microscopia Eletrônica , Peso Molecular , Óvulo/ultraestrutura , Ouriços-do-Mar
11.
Dev Biol ; 135(1): 111-23, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2767332

RESUMO

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.


Assuntos
Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Membranas Intracelulares/ultraestrutura , Óvulo/ultraestrutura , Ouriços-do-Mar/ultraestrutura , Actinas/análise , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Gema de Ovo/análise , Eletroforese em Gel de Poliacrilamida , Lipídeos de Membrana , Proteínas de Membrana/análise , Microscopia Eletrônica , Peso Molecular , Óvulo/análise , Fosfolipídeos/análise , Ouriços-do-Mar/análise
12.
Dev Biol ; 132(1): 113-29, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2492960

RESUMO

A monoclonal antibody (MAb No. 25-16), raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus, has been used to identify a previously uncharacterized CV-derived polypeptide component of the sea urchin fertilization envelope (FE). MAb No. 25-16, an IgG1, bound to a group of proteins with Mr approximately 200,000 on immunoblots of CVs. This same group of proteins also was detected in fertilization product and in soft FEs prepared from early embryos, indicating that the antigen is released at fertilization by CV exocytosis and becomes incorporated into the FE. The multiple components recognized by MAb No. 25-16 apparently did not result from proteolysis during sample preparation or differential N-linked glycosylation. No simplification of the SDS-gel or immunoblot patterns was observed when samples of fertilization product or cell surface complex were prepared in the presence of a cocktail of protease inhibitors; nor was a change in mobility of any of the antigen forms detected following treatment with endoglycosidase F. Upon partial denaturation and reduction of the protein by incubation at room temperature in the presence of SDS and dithiothreitol, the antigen was shown to undergo a decrease in relative mobility on SDS-PAGE. Complete reduction and denaturation, by boiling in dithiothreitol-containing SDS sample buffer or by an on-blot reduction technique, resulted in loss of the epitope. The protein component recognized by MAb No. 25-16 underwent a striking increase in mobility on SDS-PAGE after chelation of calcium ions with EGTA. Immunogold labeling on thin sections of unfertilized eggs revealed that the antigen is located in the spiral lamellar cores of all CVs. In fertilized eggs, fixed 5 min after insemination, the antigen was detected in the FE. Based on these biochemical and immunological data, we suggest that the antigen recognized by MAb No. 25-16 is released exocytotically from the CVs into the perivitelline space at fertilization and becomes incorporated into the FE. The abundance of this antigen suggests that it may function as a structural component of the FE.


Assuntos
Proteínas do Ovo/análise , Fertilização , Ouriços-do-Mar/análise , Zigoto/análise , Animais , Anticorpos Monoclonais/análise , Western Blotting , Cálcio/farmacologia , Grânulos Citoplasmáticos/análise , Imunofluorescência , Glicosídeo Hidrolases/farmacologia , Temperatura Alta , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Microscopia Eletrônica , Peso Molecular , Oxirredução , Inibidores de Proteases/farmacologia
13.
Biochem J ; 237(3): 853-8, 1986 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-3026334

RESUMO

A new mechanism for activation of the proactivator of procollagenase [Vater, Nagase & Harris (1983) J. Biol. Chem. 258, 9374-9382] has been found. Collagenolytic and other proteolytic enzyme activities in the medium of cultured rabbit synovial fibroblasts were found to be activated by a new mechanism: short-term incubation at 37 degrees C performed in the presence of EGTA followed by replacement of Ca2+ during enzyme assay. The crucial event in procollagenase activation is the production of a functional activator enzyme. Activation of procollagenase in the culture medium did not occur when proactivator was removed by immunoprecipitation. Proteolytic activity of proactivator was fully activated, whereas procollagenase alone could not be activated by the same sequence. EGTA treatment of the culture medium at 0 degrees C did not result in enzyme activation if Ca2+ was replaced before incubation at 37 degrees C. Certain other bivalent metal ions (e.g. Sn2+, Cd2+, Zn2+ and Mn2+) could substitute for Ca2+ to stabilize the proactivator as a zymogen and therefore prevent the appearance of proteolytic activity in culture medium. Isolation of proactivator and procollagenase from EGTA-treated radiolabelled culture medium by immunoprecipitation and subsequent analyses by fluorography revealed that a time-dependent proteolysis of both molecules occurred after replacement of Ca2+ and incubation at 37 degrees C. However, comparison of enzyme activity with fluorographic analyses showed that the maximal activation of both enzymes was achieved before any detectable decrease in Mr. The results suggest that the activation of proactivator and the subsequent activation of procollagenase may be initiated by conformational changes in structure of the proactivator molecule produced by removal of stabilizing bivalent metal ions.


Assuntos
Colagenases , Ácido Egtázico/farmacologia , Precursores Enzimáticos/metabolismo , Colagenase Microbiana/metabolismo , Animais , Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Imunoglobulina G/imunologia , Conformação Proteica , Coelhos
14.
Nature ; 315(6022): 777-8, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-3159967

RESUMO

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) is present in many mammalian tissues, and its important physiological protein substrates are only now beginning to be identified. A useful advance in identifying these intracellular substrates has been the recognition that the kinase is the receptor for phorbol esters, which stimulate phosphotransferase activity. Phorbol ester-induced changes in protein phosphorylation in intact cells may thus be taken, in part, as a probable indication of protein kinase C activation. The many cellular effects of phorbol esters include the stimulation of glucose uptake, although the response of glucose uptake to phorbol esters appears to be complex, apparently varying in response time and requirement for protein synthesis. Such observations prompted us to explore one possible explanation for the alteration of glucose uptake, namely, phosphorylation of the glucose transporter by protein kinase C. We report here that incubation of purified human erythrocyte glucose transporter with rat brain protein kinase C results in the phosphorylation of a protein of relative molecular mass (Mr) 50,000-60,000 which has subsequently been identified as the glucose transporter by specific immunoprecipitation with a monoclonal antibody. Immunoprecipitation of membrane proteins from 32P-labelled human erythrocytes revealed a phorbol ester-stimulated phosphorylation of the transporter. This covalent modification of the glucose transporter may thus, in part, underlie the ability of phorbol esters and certain hormones to stimulate glucose uptake.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Quinases/metabolismo , Eritrócitos/metabolismo , Humanos , Peso Molecular , Proteínas de Transporte de Monossacarídeos , Fosforilação , Proteína Quinase C
15.
Biochem J ; 214(2): 281-8, 1983 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-6311179

RESUMO

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.


Assuntos
Colagenases , Precursores Enzimáticos/metabolismo , Furanos/farmacologia , Colagenase Microbiana/metabolismo , Monensin/farmacologia , Membrana Sinovial/enzimologia , Animais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/biossíntese , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Colagenase Microbiana/antagonistas & inibidores , Colagenase Microbiana/biossíntese , Coelhos , Membrana Sinovial/efeitos dos fármacos , Tunicamicina/farmacologia
16.
J Biol Chem ; 258(15): 9374-82, 1983 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-6308006

RESUMO

Procollagenase and a latent activator of procollagenase were found in culture medium from rabbit synovial fibroblasts stimulated either with crystals of monosodium urate monohydrate or with phorbol myristate acetate. Procollagenase (pI 6.8) and activator (pI 5.48) could be separated by isoelectric focusing. Both procollagenase and the latent activator were purified to electrophoretic homogeneity by immunoadsorption chromatography. The activator was found to exist as a doublet of Mr = 53,400 and 51,900, with conversion to a lower Mr form upon treatment with either trypsin or 4-aminophenylmercuric acetate. In the absence of activator, purified procollagenase was not activated either by trypsin or by 4-aminophenylmercuric acetate, although both agents were capable of converting procollagenase to a lower Mr form. Activation of crude procollagenase was inhibited by immunoprecipitation of latent activator prior to addition of trypsin. Procollagenase activation increased with increased concentration of activator and with increased time of incubation with activator. The activator itself had no collagenolytic activity. The activation did not result in an apparent decrease in Mr of the procollagenase molecule as seen on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both high and low Mr procollagenase (trypsin-treated) could be activated by the activator. Purified activator, but not purified procollagenase, contained latent proteolytic activity against azocasein, gelatin, and reduced carboxymethylated bovine serum albumin substrates.


Assuntos
Colagenases , Precursores Enzimáticos/metabolismo , Colagenase Microbiana/metabolismo , Proteínas/isolamento & purificação , Líquido Sinovial/citologia , Animais , Cromatografia de Afinidade , Ativação Enzimática , Fibroblastos/enzimologia , Imunodifusão , Colagenase Microbiana/imunologia , Peso Molecular , Coelhos
18.
J Biol Chem ; 256(23): 11951-4, 1981 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-6271768

RESUMO

mRNA extracted from rabbit synovial fibroblasts which had been induced to produce large amounts of collagenase (EC 3.4.23.7) by urate crystals was translated in a cell-free wheat germ system. Collagenase was identified by immunoprecipitation using mono-specific antibody to rabbit synovial collagenase. In the absence of microsomal membranes, a single precursor with Mr = 59,000 was synthesized. This polypeptide was susceptible to proteolytic degradation. In the presence of canine pancreatic microsomes, the nascent protein was processed to a polypeptide with Mr = 57,000 (identical in mobility on sodium dodecyl sulfate-gel electrophoresis to the major latent collagenase secreted from cells) and was protected from tryptic digestion unless a detergent was used to disrupt the membranes. In addition to Mr = 57,000 material, cells secreted immunologically reactive latent collagenase with Mr = 61,000. High molecular weight collagenase was separated from Mr = 57,000 species by binding to concanavalin a-Sepharose, suggesting that this enzyme was a product of post-translational glycosylation. Both latent enzymes were activated by trypsin and human plasma kallikrein to Mr = 45,000 and 49,000. The evidence indicates that rabbit synovial fibroblast collagenase is synthesized and secreted as a single polypeptide zymogen, not as an enzyme-inhibitor complex.


Assuntos
Precursores Enzimáticos/genética , Colagenase Microbiana/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Líquido Sinovial/enzimologia , Animais , Células Cultivadas , Fibroblastos/enzimologia , Colagenase Microbiana/isolamento & purificação , Peso Molecular , Coelhos
19.
Coll Relat Res ; 1(6): 527-42, 1981 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6286237

RESUMO

Collagenase produced by monolayer cultures of rabbit synovial fibroblasts was purified from serum-free culture medium. Antiserum to the pure preparation was raised in sheep. The IgG fraction has been isolated and characterized and shown to be specific for collagenase. The antibody is capable of precipitating and inhibiting the activity of both latent and activated rabbit synovial fibroblast collagenase. Cross-reactivity with human antigen from rheumatoid synovial cell culture medium is described.


Assuntos
Anticorpos/isolamento & purificação , Colagenase Microbiana/imunologia , Líquido Sinovial/enzimologia , Animais , Células Cultivadas , Reações Cruzadas , Fibroblastos/enzimologia , Imunodifusão , Imunoeletroforese Bidimensional , Peso Molecular , Coelhos
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