Abnormal Glycosylation of Duodenal Membranous Alkaline Phosphatase Induced by Cysteamine in Rats

Abstract

The membranous and soluble isoenzymes of alkaline phosphatase (AP-ase) were isolated from the duodenal mucosal cells in control and cysteamine-treated rats. Both the cytosolic and membranous isoenzymes of the AP-ase were drastically inhibited by a single subcutaneous injection of cysteamine-HCl, a potent duodenal ulcerogen. However, cysteamine did not change the isoenzyme distribution between the two compartments of the duodenal mucosal cells. Membranous isoenzyme of duodenal AP-ase in control rats was separated by concanavalin A (Con A) affinity chromatography into three fractions: unbound (65%), weakly bound (11 %) and strongly bound (24%). The strongly bound fraction disappeared completely from duodenal mucosal cells of cysteamine-treated rats before any visible signs of ulcer formation. Since the strongly bound fraction of AP-ase contains high mannose-type sugar chains, we concluded that the ulcerogen affected the initial mannosylation (or glypiation) of membranous AP-ase which took place in the rough endoplasmic reticulum of the mucosal cells. In control rats, the Con A fractions of the membranous AP-ase were separated by gel filtration as tetramers of M_r 420 and 340 kD and dimers of M_r 185-200 kD. In cysteamine-treated rats, neither the strongly bound tetramers of 340 kD nor dimers of 185 kD were found by gel filtration. However, cysteamine did not change the total tetramer:dimer ratio. Judging by the decrease of V_max values at the pH optimum, the different molecular forms of the AP-ase are selectively sensitive to cysteamine inhibition. The kinetic data show that the type of the tissue-specific inhibition by cysteamine is different for dimeric and tetrameric forms of the membranous AP-ase. It is of an ‘uncompetitive’ type for a tetrameric form and of a ‘non-competitive’ type for dimeric forms.

Microheterogeneity of Rat Serum Alkaline Phosphatase in Fasting State: Characterization of Two Duodenal Alkaline Phosphatase Glycoforms

“…Hence, the aim of the present study was to isolate and characterize the rat serum APase Con A glycoforms (taken in the fast ing state) by their thermostability, L-phenylalanine inhi bition and kinetic properties. Contrary to three sets of glycoforms that we found in duodenal mucosal cells [8][9][10], the results presented here reveal only two glycoforms of the intestinal APase in the serum of normal fasted rats, one weakly and the other strongly bound to Con A-Sepharose. Both intestinal APase glycoforms in the serum of rats are composed only of dimeric molecules of APase, as opposed to duodenal mucosal cells, where we found tetrameric and dimeric molecular glycoforms with the same binding affinity for Con A-Sepharose [8][9][10].…”

“…Figure 1 compares the Con A-Sepharose elution profiles of APase from both duodenal mucosal cells (membranous and soluble fractions) and the serum of fasted rats. The results confirmed our earlier findings [8][9][10], showing that the three APase glycoforms (un bound, I; weakly bound, II and strongly bound, III) were obtained from both the membranous and soluble frac tions of duodenal mucosal cells, while only two glyco forms were found in the serum ( fig. 1,2).…”

“…The 105,000# pellet was used as a source of the membranous and the 105,000 g superna tant as a source of the soluble APase. Further purification of the membranous and soluble APase was performed by the methods described previously [8][9][10],…”

“…divided into small portions and stored at -1 8°C for longer time intervals, as suggested by Trepanier et al [12]. APase fractions under nondcnaturating conditions was performed as described earlier [10]. Prior to application, all samples were pre treated with 2% SDS at 48°C for 5 min.…”

“…For in stance, it was shown by us recently that the Con A-Sepharose strongly bound glycoform disappeared completely from the membranous and cytosolic fraction of the duo denal mucosal cells in cysteamine-treated rats before any visible signs of the duodenal ulcer formation [9,10]. After fat feeding, the Con A weakly bound glycoform of APase increased several times in the membranous and cytosolic fractions of mucosal cells of the duodenum and in the serum of rats [11].…”