Studies on the mechanism of activation of aminopeptidases in intestinal epithelial cells cultured with peptides.

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07660167
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 食品科学・栄養科学
• Research Institution: Hiroshima University
• Principal Investigator: NISHIMURA Toshihide Hiroshima University, Faculty of Applied Biological Science, Associate Professor, 生物生産学部, 助教授 (70180643)
• Project Period (FY): 1995 – 1996
• Keywords: Aminopeptidase / Intestinal epithelial cell / Peptide / myofiblillar proteins / Wheat gluten / Cell growth / Cell proliferation

Research Abstract

Activities of intestinal aminopeptidases have been shown to be greater in rats fed a high protein diet than in rats fed a low protein diet. This study was performed to clarify the mechanism of their activation by feeding a high protein diet.
The culture of intestinal epithelial cells (IEC-6) in the medium containing myofiblillar protein hydrolysate by enzymatic treatment promoted their growth after 5 days. The specific activities of intracellular aminopeptidases in IEC-6 cells were also activated after 5 days compared with control cells cultured without its hydrolysate. The aminopeptidase activity toward Ala-beta-naphthylamide (Ala-NA) in cells cultured with hydrolysate for 7 days was about 1.4 times that in cells cultured without hydrolysate. The intracellular aminopeptidases extracted from cells were separated on DEAE-cellulose column chromatography. The adsorbed aminopeptidases were eluted by linear gradient of NaCl concentration (0 to 0.30 M) in 10 mM Tris-HCl buffer (pH7.2), at lea st five aminopeptidases being detected. The activity of an aminopeptidase eluted at 0.133 M NaCl was shown to be significantly greater in the cells cultured with the hydrolysate than in the cells cultured without hydrolysate.
The culture of intestinal epithelial cells (IEC-18) in the medium containing wheat gluten hydrolysate by enzymatic treatment also activated the specific activities of their intracellular aminopeptidases after 2 to 3 days. The aminopeptidase activity toward Leu-NA in cells cultured with hydrolysate for 2 days was about 1.5 times that in cells cultured without hydrolysate. The comparison of the pattern on anion-exchange chromatography of the extracts from the cells cultured with and without the hydrolysate revealed that he activity of an aminopeptidase eluted at 0.12 M NaCl was significantly great in the cells cultured with the hydrolysate. This aminopeptidase possessed an optimal pH of 6.5 toward Leu-methylcoumarylamide, its molecular weight being about 100,000 on SDS-PAGE and gel filtration.