[Alkaline phosphatase of the rat small intestine--purification of the enzyme and its physiological significance].
It has been known that feeding of high-fat diets increases in intestinal and serum alkaline phosphatase (AP) activity in rats. This phenomenon is regarded as an indirect evidence for a participation of intestinal AP (I-AP) in regard to fat absorption. This study was performed to clarify the role of I-AP in the fat absorption. The results can be summarized as follows: I-AP was purified about 1,800-fold from rat intestinal mucosa by n-butanol extraction, acetone precipitation, and serial chromatographies with L-Phe-Sepharose, Sephadex G-200, and DEAE-Sephadex. The purity of the enzyme was about 90-95% as judged by SDS-PAGE. On SDS-PAGE under nonreducing conditions, the purified I-AP gave two bands corresponding to a Mw of 140,000 (major) and 120,000 (minor), respectively. Under reducing conditions, the enzyme dissociated into subunit with a Mw of 60,000. The isoelectric point of I-AP was 4.6, and the Km value for p-nitrophenylphosphate was estimated to be 0.7 mM. The enzyme activity was 50% inhibited by 4mM L-phenylalanine. A simple method for measuring I-AP isozyme specifically in rat serum was developed by the use of anti I-AP antibody-bound filter paper discs. This method has revealed that the I-AP activity normally accounts for about 80% of the total AP activity in rat serum. The I-AP activity in rat serum decreased to less than one-half the initial value after 24hr-fasting. After 48hr-fasting rats were fed with normal diets, the I-AP activity returned to normal level in about 12hr. Fasting rats were fed with 30 Cal of either corn oil or sucrose. In only the fat-fed rats, the serum I-AP activity increased gradually for 8hr. Rats were then allowed free access to the normal diets, the serum I-AP activity increased in both groups almost at the same rate. There was a great variability of serum and tissue I-AP activities in each rats. An inversed relationship was present between the tissue and serum I-AP activities in each rats. This phenomenon was more significant in jejunum than in duodenum. The meaning of these results will be discussed.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography; D004041:Dietary Fats / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007408:Intestinal Absorption; D007421:Intestine, Small / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D051381:Rats
D004041:Dietary Fats; D000469:Alkaline Phosphatase