Glutathione and gamma-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa.

Abstract

Villus tip cells and crypt cells of rat jejunal mucosa were separated bythe planing procedure of Imondi et a]. and were studied with respect to their activities of the enzymes of the 'y-glutamyl cycle and glutathione content.The villus tip cells exhibit much higher y-glutamyl transpeptidase activities than do the crypt cells; thus, y-glutamyl transpeptidase appears to be a villus-specific enzyme. y-Glutamyl cyclotransferase and the enzymes required for glutathione synthesis are not specifically localized to In the present work, the micrometer planing technique devised by Imondi et al. (7) was used to separate the epithelial cells of rat jejunum. The intracellular concentration of glutathione and the activities of enzymes involved in glutathione metabolism were examined; two of these ('y-glutamyl cysteine synthetase, glutathione synthetase) catalyze the synthesis of glutathione from its constituent amino acids, and two (y-glutamyl transpeptidase, y-glutamyl cyclotransferase) are involved in the major degradative pathway of glutathione metabolism (17). The uptake of amino acids by the isolated crypt and villus cells was also studied. Methods. Male Sprague-Dawley rats (250-300 g) were housed in stainless steel cages with wire mesh bottoms; the animals were fed ad lib. on standard laboratory rat chow. Groups of rats were fasted for 24 or 48 hr; some were fasted for 24 hr, and then fed ad lib. with cane sugar cubes. Other rats were fasted for 24 hours then fed ad lib. a protein mixture prepared by mixing 8.0 g of gelatin (Knox) and 10 g of casamino acids (Difco) with 400 ml of water.The animals were sacrificed by decapitation and their small intestines were excised. The duodenum was removed and the first 18 cm of the jejunum was cut into three sections, each of which was mounted on the tissue planing device and planed at 125 ,um depths according to the method of Imondi et al. (7). The fractions were quick-frozen in petri dishes placed on dry ice. Five fractions were obtained from the animals fed ad lib. For the enzyme studies the cells were thawed and homogenized in five volumes (weight/volume) of 10 mM MgCl2, 10 mM Tris-HCl (pH 7.6). The homogenate was layered on 18% sucrose containing 10 mM MgCl2 and 10 mM Tris-HCl (pH 7.6) and centrifuged at 100,000 X g to sediment y-glutamyl transpeptidase. y-Glutamyl transpeptidase was assayed after diluting the pellet to an appropriate volume with 0.1 M Tris-HCl (pH 8.0). The other enzymes were assayed in the supernatant collected at the top of the sucrose layer. Protein was determined by the method of Lowry (20) using bovine serum albumin as standard. Thymidine kinase was assayed by the method of Klemperer and Haynes (21) except that thymidine and thymidine 5'-phosphate were separated by ascending paper chromatography in tertiary butanol:methylethylketone:water:formic acid (44:44:11:0.26; vol/vol) (22). -y-Glutamyl transpeptidase was assayed with L-y-glutamyl-p-nitroanilide and glycylglycine 420

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