ラット小腸平滑筋におけるLecithinおよびLysolecthinの水解に関する研究
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Studies on enzymatic hydrolysis of lecithin and lysolecithin in the small intestinal smooth muscle was carried out in comparison with that in the small intestinal mucosa. Hydrolytic activity was routinely determined by incubation of a substrate, 32P-lecithin or 32P-lysolecithin, and 700 × g homogenate of the small intestinal smooth muscle or mucosa as the enzyme source, in a 0.1 M Tris-maleate buffer solution (pH 7.2) at 37 ℃. 1. Hydrolysis of lecithin : Activity of lecithin hydrolysis was observed in the small intestinal smooth muscle as well as in the mucosa. Specific activity of the former was 0.6 (lecithin-P/mg of 700 × g supernatant protein/h) and the value corresponded to 3/5 of that in the latter. A slight difference on optimum pH was found in the two layers, namely, 7.2 in the former and 7.8 in the latter. Effect of Ca++ on lecithin hydrolysis was scarcely noticed in a range of 1 ～ 10 mM in the smooth muscle. An addition of EDTA did not affect the hydrolysis likewise. The same result was seen in the mucosa. The hydrolysis of lecithin was enhanced by the presence of various concentrations of deoxycholate in the former as well as in the latter. Two peaks were found at 1 mM and 5 mM respectively. Activity at 1 mM of deoxycholate was 1.5 times higher than that at 0 mM of deoxycholate and 3 times at 5 mM. Furthermore, an addition of deoxycholate in excess of 2.5 mM resulted in an accumulation of lysolecithin. In the absence of deoxycholate it was difficult to detect an increase of amount of lysolecithin. Intracellular distribution of lecithin hydrolytic activity was in the descending order of cell sap fraction ＞ microsomal fraction ＞ mitochondrial fraction in the smooth mussle, while in the descending order of microsomal ＞ cell sap ＞ mitochondrial in the mucosa. However, the value of the specific activity of each subfraction was in the following order : microsomal ＞ mitochondrial ≫ cell sap in the former, while mitochondrial ＞ microsomal ≫ cell sap in the latter. 2. Hydrolysis of lysolecithin: Activity of lysophospholipase was clearly demonstrated in the smooth muscle as well as in the mucosa. The specific activity was 340 (lysolecithin-P/mg of 700 × g supernatant protein/h) and the value corresponded to about 4/5 of that in the mucosa. Optimum pH of lysophospholipase was shown to be 6.8 in the smooth muscle. This was also the case in the mucosa. An addition of Ca++ showed a slight inhibitory effect on the enzymatic activity in the smooth muscle. It was found that deoxycholate clearly inhibited hydrolysis of lysolecithin in the smooth muscle as already found in the mucosa. Only about 5% of the original activity was observed in the presence of 10 mM or more of deoxycholate. Intracellular distribution of lysophospholipase activity was in the following order, cell sap ＞ microsomal ≫ mitochondrial in the smooth muscle, while cell sap ≒ microsomal ≫ mitochondrial in the mucosa. A comparison of each specific activity was run in the three subcellular fractions and it was shown that the following, order microsomal ＞ cell sap ≒ mitochondrial, was present in the two layers.
- 1976-10-30
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