RESUMO
An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase. A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length. Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity. In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity. Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME. DME effectively inhibited only the GmCK2-encoded choline kinase activity. Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme. These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.
Assuntos
Colina Quinase/genética , Glycine max/genética , Isoenzimas/genética , Sequência de Aminoácidos , Sequência de Bases , Colina Quinase/metabolismo , Clonagem Molecular , DNA Complementar , Escherichia coli/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Glycine max/enzimologia , Especificidade por SubstratoRESUMO
Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.
Assuntos
Diacilglicerol Colinofosfotransferase/genética , Genes de Plantas/genética , Glycine max/genética , Proteínas de Plantas/genética , Proteínas de Soja , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cistina Difosfato/análogos & derivados , Cistina Difosfato/farmacologia , DNA Complementar/genética , Diacilglicerol Colinofosfotransferase/efeitos dos fármacos , Diacilglicerol Colinofosfotransferase/metabolismo , Escherichia coli/genética , Etanolaminofosfotransferase/deficiência , Etanolaminofosfotransferase/genética , Etanolaminas/farmacologia , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Glycine max/enzimologia , Transcrição GênicaRESUMO
The amino terminal sequences of the kappa light chains from SAMM 368 IgG2b and IgA have been determined. The two chains belong to different kappa subgroups as indicated by 10 amino acid differences among the first 23 residues. An additional three residue differences were demonstrated in the sequence including the first complementarity region. These results indicate that single cells of SAMM 368 produce two unique kappa-chains.
Assuntos
Imunoglobulina A/análise , Imunoglobulina G/análise , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/análise , Cadeias kappa de Imunoglobulina/análise , Plasmócitos/imunologia , Sequência de Aminoácidos , Animais , CamundongosAssuntos
Encefalomielite Autoimune Experimental/genética , Doença Aguda , Fatores Etários , Animais , Antígenos , Doença Crônica , Encefalomielite Autoimune Experimental/imunologia , Feminino , Adjuvante de Freund , Cobaias , Técnicas In Vitro , Masculino , Coelhos , Fatores Sexuais , Especificidade da Espécie , Medula EspinalRESUMO
Major variables which determine the induction and severity of adoptive autoimmune encephalomyelitis are the age and strain of the animal, and the amount of killed mycobacteria in the adjuvant. Control of these factors results in consistent production of this disease in high incidence and in severe form. The pathologic changes in the central nervous system can be correlated with the clinical disease. Maturity of the target tissues in the central nervous system of the newborn appears to be an important factor which distinguishes the response of the guinea pig from that of other species.
