Abstract:The ALP activities and properties of rat intestine cut into 20 segments were examined, and we were able to demonstrate that the ALP activity of upper intestine is high compared to that of lower intestine. This result coincided with those of other reports. However, we newly clarified that there is an ALP isoenzyme found in the lower intestine which can be inhibited by L-homoarginine. The molecular weight of the ALP isoenzyme was 136 kDa. In addition, it was clarified that there are several isoenzymes from upper to lower intestine. This study demonstrates that there exist isoenzymes, which are inhibited by LHArg, in the intestine which are similar to the isoenzymes in the liver, bone and kidney. Key words: alkaline phosphatase (ALP), intestine, isoenzyme, L-homoarginine (L-HArg), rat by a high fat diet, ALP has been thought to be related to the absorption of fatty acid, carbohydrate, amino acid and protein from the intestine.We have found that the activities of ALP in the intestine or in serum are changed depending on the feeding conditions and have had an interest in factors which modulate the quantity of ALP protein and the activity of ALP. In a study on inhibitory effects of various compounds, it was reported that intestinal ALP is specially inhibited by L-phenylalanine (L-Phe), but not by L-homoarginine (L-HArg) [5,15]. However, in this study we found that there are some intestinal ALP isoenzymes inhibited by L-HArg. The purpose of this study was to demonstrate the properties of intestinal ALP isoenzymes by polyacrylamide gel electrophoresis (PAGE).

SummaryChanges of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electro phoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoargi nine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

Abstract:Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-Dglucosamine (GlcNAc)-Sepharose 6B column chromatography. It was concluded that this method is useful for clinical examination in the rat.