RESUMO
We have previously identified a rice gene encoding a 27 kDa protein with a single Ca2+-binding EF-hand and a putative membrane anchor. We report here similar genes termed caleosins, CLO, in other plants and fungi; they comprise a multigene family of at least five members in Arabidopsis (AtClo1-5). Northern hybridization demonstrated that AtClo2-4 mRNAs levels were low in various tissues, while AtClo1 mRNA levels were high in developing embryos and mature seeds. Analysis of transgenic Arabidopsis plants expressing the GUS reporter under control of the AtClo1 promoter showed strong levels of expression in developing embryos and also in root tip cells. Antibodies raised against AtCLO1 were used to detect caleosin in cellular fractions of Arabidopsis and rapeseed. This indicated that caleosins are a novel class of lipid body proteins, which may also be associated with an ER subdomain.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Brassica/genética , Proteínas de Ligação ao Cálcio/metabolismo , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The composition of the two major lipidic organelles of the tapetum of Brassica napus L. has been determined. Elaioplasts contained numerous small (0.2-0.6 micron) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid. This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies. The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins. Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins. The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins. The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol >> cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts. These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains.
Assuntos
Brassica/química , Lipídeos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Dados de Sequência Molecular , Pólen/químicaRESUMO
During streptokinase (SK) recovery by cross-flow microfiltration (CFMF), a loss of activity of 14.4% was observed, after the initial volume was concentrated 8-fold (VCF = 8.0); 51.5% of the activity was recovered in the filtrate and 34.1% remained in the retentate. Immunological experiments using polyclonal antibodies against SK have demonstrated that SK activity loss during CFMF processes could be related to denaturation of SK, forming molecules of lower or no activity. Accumulation of denatured SK in the retentate suggests that denaturation could be the results of an aggregation phenomenon (as has been demonstrated by light scattering and chromatographic studies), leading to the formation of protein aggregates that are retained by the microfiltration (MF) membrane, affecting the fouling phenomenon and the concentration in the retentate of permeable molecules.
Assuntos
Estreptoquinase/isolamento & purificação , Cromatografia em Gel , Dicroísmo Circular , Densitometria , Eletroforese em Gel de Poliacrilamida , Fermentação , Testes de Precipitina , Desnaturação Proteica , Espalhamento de Radiação , Streptococcus/enzimologia , Estreptoquinase/química , UltrafiltraçãoRESUMO
Streptokinase (SK) recovery from streptococcal fermentation broth by cross-flow microfiltration has been studied. Recovery of SK in the filtrate, independent of the volumetric concentration factor, is approximately two-fold lower than the initial SK activity in the fermentation broth; moreover, the SK activity in the retentate increase during the process, reaching a concentration factor of 2.73. These results show that the membrane works more as an ultrafiltration membrane, with rejection of S = 0.6, than as a microfiltration membrane. Under filtration conditions, the membrane permeation rate decreased with time. This decreased could be explained by deposition and interaction of material onto/with the membrane resulting in the concentration of permeable products. Studies of the individual concentration factors for the main streptococcal exocellular proteins, indicate clearly that the concentration of the proteins during the microfiltration process is independent of the size of the proteins, suggesting that other factors, such as charge and hydrophobicity, along with concentration-polarization, should be taken also into account for the understanding of this phenomenon.
