Single Band In SDS-polyacrylamide Gel Electrophoresis Research Articles

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn2+ and Cu2+ and inactivated by Fe3+. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.

Purification of 2,3-oxidosqualene: .BETA.-amyrin cyclase from pea seedlings.

2, 3-Oxidosqualene : β-amyrin cyclase (E.C.5.4.99) was purified from pea seedlings in eight steps as a soluble and homogeneous enzyme. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular weight of 35 K Da and had a Km value of 50 μM. It was demonstrate that β-amyrin cyclase had a different molecular weight from that of cycloartenol cyclase, and these two enzymes were responsible for the dramatic alteration in triterpenoid and steroid biosynthesis during germination.