RESUMO
The microtubule-associated proteins 1B and 1A are synthesized as polyprotein precursors which are rapidly cleaved to give rise to heavy and light chains constituting the respective microtubule-associated protein 1B or microtubule-associated protein 1A complex. To identify domains necessary for precursor processing, we expressed microtubule-associated protein 1B deletion mutants in fibroblasts and monitored proteolytic cleavage of the precursor proteins by immunoblot analysis. We found that a novel hydrophilic, proline-rich 45 amino acid domain containing the cleavage site is necessary and sufficient for processing. This domain is conserved in microtubule-associated protein 1A. Additional sequences in the N-terminal half of the heavy chain contribute to the efficiency of cleavage.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Endopeptidases/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Precursores de Proteínas/genética , Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta.
Assuntos
Glândulas Mamárias Animais/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Animais Geneticamente Modificados , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/citologia , Humanos , Lactação/metabolismo , Masculino , Espectrometria de Massas , Leite/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Especificidade de Órgãos , Células PC12 , RNA Mensageiro/análise , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain-induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain-light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Células 3T3 , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Endopeptidases/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/fisiologia , Dados de Sequência Molecular , Células PC12 , RatosRESUMO
Recently, the concept of microtubule-associated protein 1B as an intracellular 2460 amino acid protein was challenged by the suggestion that only the N-terminal 1022 codons are utilized and encode the core protein of the extracellular proteoglycan claustrin (Burg and Cole (1994) J. Neurobiol. 25, 1-22). We expressed this N-terminal MAP1B fragment in tissue culture cells and found that it bound to microtubules and was not localized in the extracellular matrix. In addition, epitope mapping demonstrated that MAP1B consisted of more than 1022 amino acids and that the reported cDNA of claustrin is incomplete.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Sulfato de Queratano/genética , Proteínas Associadas aos Microtúbulos/genética , Proteoglicanas/genética , Células 3T3 , Animais , Encéfalo/citologia , Proteoglicanas de Sulfatos de Condroitina/química , Sulfato de Queratano/química , Lumicana , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Ratos , Células Tumorais Cultivadas/metabolismoRESUMO
Two polyclonal antibodies were raised against the gamma 1-subunit of gamma-aminobutyric acidA (GABAA) receptors. One of these antibodies could be used for the immunopurification of GABAA receptors containing gamma 1-subunits. These receptors exhibited [3H]muscimol and [3H]flunitrazepam binding, and their benzodiazepine binding properties were dramatically different from those of receptors precipitated by anti-gamma 2- or anti-gamma 3-antibodies. Western blot analysis of the immunopurified GABAA receptors indicated that the gamma 1-subunit exhibits an apparent molecular mass of 45-51 kDa. Furthermore, in addition to gamma 1-subunits, beta 2/3-, alpha 1-, alpha 2-, alpha 3-, and alpha 5-subunits could be detected in immunoaffinity column eluates from total brain. In contrast, gamma 2- or gamma 3-subunits could not be identified in GABAA receptors immunopurified by anti-gamma 1-antibodies. Similarly, gamma 1- and gamma 3-subunits or gamma 1- and gamma 2-subunits could not be identified in GABAA receptors purified by anti-gamma 2- or anti-gamma 3-antibodies, respectively. These data seem to indicate that GABAA receptors contain only a single type of gamma-subunit.
Assuntos
Química Encefálica , Receptores de GABA-A/isolamento & purificação , Animais , Especificidade de Anticorpos , Sequência de Bases , Western Blotting , Cromatografia de Afinidade , Clonazepam/metabolismo , Ensaio de Imunoadsorção Enzimática , Flumazenil/metabolismo , Flunitrazepam/metabolismo , Técnicas Imunológicas , Dados de Sequência Molecular , Peso Molecular , Muscimol/metabolismo , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Ratos , Receptores de GABA-A/classificação , Receptores de GABA-A/imunologia , Receptores de GABA-A/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Two polyclonal antibodies were raised against the gamma 3-subunit of gamma-aminobutyric acidA (GABAA) receptors. These antibodies were able to precipitate GABAA receptors from brain membrane extracts, and the precipitated receptors exhibited benzodiazepine binding properties that were dramatically different from those of receptors precipitated by anti-gamma 2 antibodies. The anti-gamma 3 antibodies were used for immunopurification of GABAA receptors containing gamma 3-subunits. Western blot analysis of the immunopurified GABAA receptors indicated that the gamma 3-subunit exhibits an apparent molecular mass of 43-46 kDa. Furthermore, in addition to gamma 3-subunits, beta 2/3-, alpha 1-, alpha 2-, alpha 3-, alpha 4-, and alpha 6-subunits could be detected in immunoaffinity column eluates from total brain and cerebellum, respectively. These data indicate that gamma 3-subunits can combine with most alpha-subunits to form a multiplicity of GABAA receptors with distinct pharmacology.