RESUMO
The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.
Assuntos
Lipase/biossíntese , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/enzimologia , Cromatografia em Agarose , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Vetores Genéticos , Glicosilação , Hexosaminidases/metabolismo , Humanos , Lipase/química , Lipase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Peso Molecular , Saccharomyces cerevisiae/genética , Transformação Genética , Triglicerídeos/químicaRESUMO
Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.