RESUMO
Calcium-dependent protein kinases (CDPKs) are found in various subcellular localizations, which suggests that this family of serine/threonine kinases may be involved in multiple signal transduction pathways. CDPKs are believed to be involved in the response of plants to low temperatures, but the precise role in the signal transduction pathway is largely unknown. Previous reports described changes in CDPKs' mRNA levels in response to cold treatment, but whether these changes are accompanied by increases in protein level and/or kinase activities is unknown. In the present study, we identify in rice (Oryza sativa L. cv Don Juan) plants a 56-kD membrane-bound CDPK that is activated in response to cold treatment. Immunoblot analysis of the enzyme preparations from control and cold-treated plants showed that the kinase level was similar in both preparations. However, both kinase and autophosphorylating activities of the enzyme prepared from cold-treated plants were significantly higher than that obtained from control plants. The activation of the CDPK is detected after 12 to 18 h of cold treatment, which indicates that the kinase does not participate in the initial response to low temperature but in the adaptative process to adverse conditions. To our knowledge, this is the first demonstration of a CDPK that is posttranscriptionally activated in response to low temperature.
Assuntos
Temperatura Baixa , Oryza/enzimologia , Proteínas Quinases/metabolismo , Western Blotting , Membrana Celular/enzimologia , Ativação Enzimática , Fosforilação , SolubilidadeRESUMO
Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.
Assuntos
Membranas/enzimologia , Ácido Mirístico/metabolismo , Oryza/enzimologia , Ácidos Palmíticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Biológico , Cálcio/farmacologia , DNA Recombinante/genética , DNA Recombinante/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Protoplastos/metabolismo , Zea mays/genéticaRESUMO
A partially active and a latent form of multicatalytic protease (MCP) were isolated from fish skeletal muscle. Both forms were inactive against protein substrates, but their activity against peptide substrates differed in one order of magnitude. The chymotrypsin-like activity of the partially active form was moderately stimulated by fatty acids and SDS, whereas its trypsin-like activity was inhibited by the same reagents. In contrast, both activities of the latent form were strongly stimulated by SDS. The chymotrypsin-like activity of the latent form was also stimulated by heating or high urea concentrations, whereas its trypsin-like activity did not change or was inhibited respectively by these treatments. These activation effects were irreversible. Pre-treatment of the latent form with SDS or urea in the absence of substrate led to its irreversible inactivation, whereas activation by pre-heating occurred in the presence or absence of substrate. These results suggest that MCP can exist in several active states with distinct properties. Studies on the distribution of MCP in fish tissues showed a much higher level of the enzyme in gonads than in any other tissue, suggesting a role of MCP in development.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/enzimologia , Animais , Cisteína Endopeptidases/isolamento & purificação , Peixes , Complexos Multienzimáticos/isolamento & purificação , Especificidade de Órgãos , Complexo de Endopeptidases do ProteassomaRESUMO
1. A latent form of multicatalytic proteinase (MCP) was purified to apparent homogeneity from white croaker muscle by DEAE-Sephacel, Mono-Q, Sephacryl S-300 and second Mono-Q chromatographies. 2. The enzyme preparation was electrophoretically and immunologically similar to MCP purified from the same source by a different method (Folco et al., 1988b, Archs Biochem. Biophys. 267, 599-605) but showed much lower chymotrypsin- and trypsin-like activities. 3. These activities responded to sodium dodecyl sulphate (SDS), urea and heat treatments in different ways: SDS stimulated both activities, urea stimulated the former and inhibited the latter and heating stimulated the former and did not affect the latter. 4. The stimulation of chymotrypsin-like activity by the three treatments was irreversible. 5. Exposure of MCP to SDS or urea in the absence of substrate rapidly inactivated it, whereas heat activation took place irrespective of the presence of substrate. 6. The stimulating effect of SDS on chymotrypsin-like activity was lost in the presence of urea. 7. These results suggest that the enzyme may be activated by different mechanisms.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Músculos/enzimologia , Perciformes/metabolismo , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Temperatura Alta , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Dodecilsulfato de Sódio/farmacologia , Ureia/farmacologiaRESUMO
When dialyzed extracts from hake (Merluccius hubbsi) skeletal muscle were chromatographed in DEAE-Sephacel, an alkaline protease (37 degrees C, pH 8.5) and a trypsin inhibitor were isolated. The enzyme showed its maximal activity against azocasein in the range of pH between 7 and 9. The protease was able to hydrolyze the trypsin substrates Bz-Arg-OEt and Tos-Arg-OMe and did not cleave the chymotrypsin substrate Bz-Tyr-OEt. The enzyme was strongly inhibited by several serine protease inhibitors, whereas inhibitors of the other types of proteases scarcely affected it. The protease was able to degrade the major contractile and cytoskeletal constituent proteins of myofibrils and to accumulate acid-soluble products. The protease activity was completely suppressed by the addition of the trypsin inhibitor isolated from the same muscle. These results indicate that hake skeletal muscle contains a trypsin-like serine protease which might be involved in the catabolism of myofibrillar proteins, as well as in the proteolytic events that take place during post mortem storage of fish muscle.
Assuntos
Peixes , Músculos/enzimologia , Serina Endopeptidases/análise , Inibidores da Tripsina/análise , Tripsina/metabolismo , Animais , Caseínas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase , Especificidade por Substrato , Inibidores da Tripsina/farmacologiaRESUMO
Proteinase I, an enzyme previously shown to be able to degrade contractile and cytoskeletal elements of white-croaker (Micropogon opercularis) myofibrils, was purified to apparent homogeneity by chromatography on DEAE-Sephacel, octyl-Sepharose CL 4B and arginine-Sepharose 4B. Its Mr was determined to be 269,000 by Sephacryl S-300 gel filtration. Under denaturing conditions, the enzyme dissociated into two subunits with Mr 20,000 and 15,500, in a molar ratio of 1.8:1. Proteinase I showed a pH optimum of 8.5. The enzyme was strongly inhibited by several serine proteinase inhibitors, whereas inhibitors of the other types of proteinases did not affect, or only scarcely affected, its activity. Several N-terminal-blocked 4-methyl-7-coumarylamide substrates having either arginine or lysine residues adjacent to the fluorogenic group were efficiently hydrolysed by the enzyme. These results indicate that proteinase I is a trypsin-like serine proteinase. The enzyme appears to be distinct from other proteinases previously described in skeletal muscle, and might be involved in the catabolism of myofibrillar proteins.
Assuntos
Peixes/metabolismo , Músculos/enzimologia , Serina Endopeptidases/isolamento & purificação , Animais , Cromatografia , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Peso Molecular , Inibidores de Proteases/farmacologia , Desnaturação Proteica , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , TemperaturaRESUMO
The extraction of white croaker skeletal myofibrils with KI rendered a residue in which a network of longitudinal and transverse filaments could be observed by scanning electron microscopy. A trypsin-like serine proteinase isolated from the same muscle was able to produce a complete and rapid disruption of the network, while major myofibrillar proteins were only slightly modified. This fact suggests that the disassembly of the cytoskeletal network may be an early event in the proteolysis of myofibrils. Desmin was not attacked by the proteinase under the assayed conditions, which indicates that some other unidentified component of the network would be the primary target of the action of the enzyme on myofibrils.
Assuntos
Citoesqueleto/ultraestrutura , Músculos/ultraestrutura , Miofibrilas/ultraestrutura , Serina Endopeptidases/metabolismo , Animais , Peixes , Microscopia Eletrônica de Varredura , Proteínas Musculares/isolamento & purificaçãoRESUMO
Proteinase II, a high-molecular-mass proteinase previously identified in white croaker skeletal muscle, was purified to apparent homogeneity by DEAE-Sephacel, phenyl-Sepharose CL 4B, and Sephacryl S-300 chromatographies. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with Mr ranging from 18,000 to 26,000 and a large subunit with a Mr 60,000. The proteinase was able to hydrolyze N-terminal-blocked 4-methyl-7-coumarylamide substrates having either an aromatic amino acid (chymotrypsin-like activity) or an arginine residue (trypsin-like activity) adjacent to the fluorogenic group. The trypsin-like activity of the enzyme was inhibited by fatty acids and sodium dodecyl sulfate, whereas the chymotrypsin-like activity was stimulated by those compounds but inhibited by nonionic and cationic detergents. Several thiol reagents inhibited both proteinase II activities. However, leupeptin and Cu2+ strongly inhibited its trypsin-like activity but only slightly affected its chymotrypsin-like activity. Dithiothreitol stimulated both activities, but at different extents and in different concentration ranges. These results suggest that the enzyme is multicatalytic, having at least two different active sites.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Músculos/enzimologia , Animais , Cromatografia/métodos , Inibidores de Cisteína Proteinase , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/farmacologia , Peixes , Hidrólise , Complexos Multienzimáticos/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma , Proteínas/análise , Especificidade por Substrato , Compostos de Sulfidrila/farmacologiaRESUMO
1. Fish skeletal muscle contains an alkaline thiol proteinase with a temperature optimum of 60 degrees C and undetectable activity below 50 degrees C. 2. The present study shows that fatty acids and sodium dodecyl sulphate (SDS) shifted the temperature-activity curve of the enzyme toward the lower temperature side. 3. All unsaturated fatty acids tested strongly stimulated proteolytic activity at 37 degrees C, whereas myristic acid was the only saturated fatty acid that produced an important degree of activation. 4. These effects could be observed at millimolar concentrations of the reagents.