Functional and Molecular Characterization of Rat Intestinal Prolidase

Abstract

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 (K m and V max ) 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 K m value of 178 M and a V max value of 601 mol · min Ϫ1 · mg protein Ϫ1 . 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. Prolidase is essential for the digestion and metabolism of protein, because it is the enzyme responsible for the metabolism of the most abundant protein digest glycyl-proline (GlyPro) and other imino-dipeptides (1). It is also important for endogenous protein metabolism and amino acid recycling, because it participates in collagen metabolism (2). It plays an important role in cellular growth because Chinese hamster cells that lack proline biosynthesis capability could grow normally as long as Gly-Pro and prolidase were present (1). Genetic deficiency of prolidase can lead to severe problems in child development, including abnormal joints, skin lesion (3), skin cancer (4), and mental retardation (5-7). In addition, abnormal prolidase activities were reported to be associated with certain side effects of drugs (8), liver cirrhosis (9), and hepatoma (10). Finally, prolidase has been targeted as a prodrug converting enzymes by various research groups, primarily for the purpose of improving systemic or cellular bioavailability of poorly absorbed drugs (11,12).It is unclear how abnormal expression of prolidase contributes to the pathogenesis of various diseases associated with its deficiency. Previous studies have not generated a definitive cause for the pathogenesis of prolidase deficiency. For example, Dolenga and Hechtman (13) showed that fibroblasts obtained from symptomatic patients ...

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