Functional and molecular characterization of rat intestinal prolidase.
Genetic deficiency of prolidase can lead to severe problems in child development, including mental retardation. However, the exact pathogenesis of the disease is unclear. To understand the enzyme's physiologic functions, we studied the regulation of rat intestinal prolidase. The results indicated that 1) the activities of intestinal prolidase and its kinetic parameters (Km and Vmax) are site-dependent; 2) the jejunal prolidase activity was the most sensitive to the dietary restriction, and the duodenal and jejunal but not colonic kinetic parameters changed with dietary restriction; 3) the pH activity profile of jejunal prolidase at 24 h postfeeding was different from that at 48 h postfeeding, whereas the inhibition profiles of prolidase were qualitatively independent of dietary restriction; and 4) old-aged rats have lower prolidase activities in the small intestine. We also purified rat intestinal prolidase I to homogeneity. The characterization study indicated that the purified rat intestinal prolidase I is fairly similar to prolidase I from other species with a molecular weight of 116,000, which consisted of two monomers, 58,000 D each. The purified prolidase I has a Km value of 178 microM and a Vmax value of 601 micromol x min-1. mg protein-1. Screening of a rat intestinal cDNA library produced a 1.8-kb fragment that encodes the rat intestinal prolidase. This enzyme has 494 deduced amino acid sequence, which is 96% or 86% identical to mouse or human erythrocyte prolidase I. This represents the first report of a successful attempt to purify and clone an intestinal prolidase and of investigation to study prolidase regulation by diet.
Hu
Ming
M
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6510, USA. minghu@wsu.edu
Cheng
Zhengqi
Z
Zheng
Lixing
L
D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004150:Dipeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004150:Dipeptidases; C022449:proline dipeptidase