RESUMO
Analysis of milk samples from a number of lactating women revealed molecular variants of bile salt-stimulated lipase (BSSL) of both lower and higher molecular mass than that commonly occurring. In contrast to previous observations, we report on individuals having only a variant of lower mass, both one of lower and one of common mass, or both one of lower and one of higher mass of the lipase. From two individuals we purified the lower molecular mass BSSL variant and characterized it. The amount of lipase in the milk of these two individuals was considerably less than average (mean of 10 women with BSSL of the most common molecular mass). The BSSL variant of lower mass showed the same bile salt activation, pH dependency, temperature stability as those most commonly occurring. We could localize the difference in mass to the large O-glycosylated repeat sequence close to the C-terminus of the protein. With respect to all characteristics studied, the BSSL variant of higher mass was also similar to that most commonly ocurring. Again, the difference in mass could be localized to the repeat region of the protein. Hence, it appears as if the repeat region, normally carrying 16 repeats of 11 amino acids each, varies in size between individuals.
Assuntos
Lipase/química , Leite Humano/enzimologia , Esterol Esterase , Aminoácidos/análise , Animais , Ácidos e Sais Biliares/farmacologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Feminino , Variação Genética , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Lactação , Lipase/genética , Lipase/isolamento & purificação , Lipase/metabolismo , Peso Molecular , Oligossacarídeos/análise , Oligossacarídeos/química , Proteínas Recombinantes/química , Sefarose/análogos & derivados , Sefarose/metabolismo , TemperaturaRESUMO
Stratum corneum chymotryptic enzyme (SCCE) is a new human serine proteinase expressed by keratinocytes in the epidermis. Its function may be to catalyze the degradation of intercellular cohesive structures in the cornified layer of the skin in the continuous sheeding of cells from the skin surface. In this work the primary substrate specificity of recombinant SCCE was determined with oxidized bovine insulin B chain as substrate. Cleavage products were separated with high performance liquid chromatography and analyzed by amino acid sequence analysis and mass spectrometry. Cleavage sites were localized to Leu 6-cysteic acid 7, Tyr 16-Leu 17, Phe 25-Tyr 26, and Tyr 26-Thr 27 in insulin B chain. It is concluded that SCCE belongs to the family of serine endoproteinases specific for amino acid residues with aromatic side chains in the P1 position.
