M. Gilles-Baillien

Intracellular Inorganic Ions and Amino-Acid Pool in the Isolated Intestinal Mucosa of the Tortoise « Testudo Hermanni Hermanni » Gmelin

AbstractIn order to study the intracellular composition of the intestinal mucosa, the first step consists of the determination of the extracellular space. A previous work (Gilles-Baillien, 1968) has shown that the extracellular space of the small intestine mucosa is close to 10% (ml/g wet weight) and is the same at the different levels of the small intestine, while it is between 6 and 10% (ml/g wet weight) in the colon mucosa. Knowing, in addition, the water content, an estimation can be given of the intracellular concentrations in Na, K, Ca, Cl, taurine, urea and amino acids, at different levels of the intestinal tract, in the single mucosa.

Alkaline phosphatase and ATPases in brush-border membranes of rat jejunum: Distinct effects of divalent cations and of some inhibitors

The effects of divalent cations and of some inhibitors on the activities of alkaline phosphatase and ATPase were examined in rat jejunal brush-border membranes (BBM) isolated by tha Ca(2+)-(BBMCa) or the Mg(2+)-precipitation method (BBMMg). Similar results were found in BBMCa and BBMMg though generally higher in BBMCa. Alkaline phosphatase activity was stimulated by 5 mM MgCl2 (30% to 44%), but not by 5 mM CaCl2 or 0.1 mM ZnCl2, at pH 9.5 or 7.4. ATPase activity was equally stimulated by 5 mM MgCl2 and by 5 mM CaCI2 (about 150%). Alkaline phosphatase activity was significantly inhibited by 1 mM vanadate, 5 mM diamox, 5.0 mM L-leucine and 1 mM theophylline. In contrast, Ca(2+)-ATPase and Mg(2+)-ATPase activities were not depressed by those alkaline phosphatase inhibitors, but were inhibited by 0.1 mM trifluoperazine (more than 70%). 0.1 mM ZnCl2 also appeared to be inhibitory to Ca(2+)-ATPase and Mg(2+)-ATPase, but not to alkaline phosphatase activity even in the presence of Ca2+ and Mg2+. These results suggest that Ca(2+)-ATPase and Mg(2+)-ATPase activities of the rat jejunal BBM are not merely manifestations of alkaline phosphatase, but rather belong to (a) distinct enzyme(s).

Ca2+-ATPase and Mg2+-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes

AbstractIn rat jejunal brush-border membranes (BBM), ATP hydrolysis activity was specifically stimulated by CaCl2 and by MgCl2, allowing to identify Ca2+-ATPase and Mg2+-ATPase activities with a broad pH optimum near 8.0. Nonspecific ATPase activity (in the absence of cations) had a pH optimum above 9.5 as alkaline phosphatase. The effects of Ca2+ and Mg2+ concentrations on ATPase activity evidenced two apparent KA for each cation. At high concentrations, a similar affinity for both cations was recorded (KA : 0.35 mM). At low concentrations, the affinity for Mg2+ was greater than for Ca2+ (KA : 0.02 mM and 0.07 mM respectively). In an attempt to differentially solubilize alkaline phosphatase and ATPase activities, eleven different detergents were assayed. They more or less successfully released Ca2+-ATPase and Mg2+-ATPase activities from BBM but the more membranes were solubilized by a detergent, the more activities were lost, suggesting a close dependence on integration in BBM. As to alkaline phosphatase...

Bound and purified alkaline phosphatase from rat duodenal and jejunal brush-border membranes: Kinetics and magnesium stimulation

Brush-border membranes from rat duodenal and jejunal mucosa were prepared by differential Ca(2+)-precipitation. Kinetical properties and Mg(2+)-stimulation of alkaline phosphatase were studied for the enzyme either bound to these membranes or purified from these membranes by liquid chromatography. With p-nitrophenylphosphate as substrate, the alkaline phosphatase apparent Km was lower in jejunum (90 microM) as compared with duodenum (160 microM), and lower for the purified enzyme (jejunum: 55 microM; duodenum: 97 microM) as compared to the bound one. In the presence of 5 mM MgCl2, the substrate affinity was in all cases decreased. For the bound enzyme Vmax was 10 times greater in duodenum compared to jejunum. 5 mM MgCl2 tripled the Vmax of the duodenal bound enzyme and increased it by 50% for the jejunal one, but a seven-fold increase was recorded for the purified enzyme at both levels of intestine. The apparent affinity for Mg2+ was similar for the bound and the free enzyme, for duodenum and for jejunum (Mg0.5: +/- 40 microM).