Microheterogeneity of rat serum alkaline phosphatase in fasting state: characterization of two duodenal alkaline phosphatase glycoforms.
Using concanavalin A-Sepharose affinity chromatography (Con A) we found that the serum of normal fasted adult rats contains two alkaline phosphatase (APase) glycoforms, one weakly bound (II) and the other strongly (III) bound to the column. Both serum APase glycoforms had an apparent molecular mass of 163 kD on Sepharose CL-6B and 118 kD on SDS-PAGE under nondenaturing conditions. We consider the molecular forms as dimeric, since monomers of 60.3 and 58.5 kD for the Con A weakly and strongly bound glycoform, respectively, were obtained. However, these two dimeric glycoforms were different in their pH optimum, affinity to p-nitrophenyl phosphate as substrate, the degree of L-phenylalanine inhibition and relative thermostability. Judging by the relative thermostability and by L-phenylalanine inhibition, it seems that both serum APase glycoforms in fasted rats are mainly of duodenal mucosal cell origin. The Con A weakly bound (II) glycoform could be derived from the cytosol, and the Con A strongly bound (III) one from both the cytosolic and membranous fractions of duodenal mucosal cells. However, in addition to the heat-stable component, the Con A strongly bound serum APase glycoform also contains a minor heat-labile and L-phenylalanine-resistant component which could be of nonspecific tissue origin since such a fraction was not discovered by us in rat duodenal mucosal cells.
Japundzic
I
I
Institute of Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia.
Kosaric
B
B
Rakic-Stojiljkovic
L
L
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000097:blood / Q000737:chemistry*; D000818:Animals; D003208:Concanavalin A / Q000378:metabolism; D004355:Drug Stability; D004386:Duodenum / Q000201:enzymology; D005215:Fasting / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology / Q000502:physiology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000187:drug effects*; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D006023:Glycoproteins; D007527:Isoenzymes; D003208:Concanavalin A; D010649:Phenylalanine; D000469:Alkaline Phosphatase