We used rat pancreatic acim as well as COS-7 cells transfected with the cloned pancreatic cholecystokinin (CCK) (8,9). In addition, reducing the incubation temperature from 3TC to 40C (7) or inhibiting cellular energy metabolism (2) abolishes the highaffinity state, but the biochemical basis of these effects is not known.Based on previous studies ofthe actions of monensin (4) Unless stated otherwise, the standard incubation solution contained 24 mM Hepes (pH 7.4), 120 mM NaCl, 7.2 mM KC1, 2.2 mM NaH2PO4, 6 mM sodium pyruvate, 7 mM sodium fumarate, 6 mM sodium glutamate, 0.5 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, 2 mM glutamine, 1% (wt/vol) albumin, 0.01% (wt/vol) trypsin inhibitor, 1% (vol/vol) essential amino acid mixture, 0.1% (wt/vol) bacitracin, and 1% (vol/vol) vitamin mixture. All incubations were performed with 10O% 02 as the gas phase.
METHODSTissue Preparation. Dispersed acini from rat pancreas were prepared according to the modifications (15) of the published procedure (16).Binding of mI-CCK-8. Binding of 125I-CCK-8 was measured as described (4,10). Dispersed acini from four pancreata were suspended in 20 ml of standard incubation solution.Abbreviations: CCK, cholecystokinin; CCK-8, CCK octapeptide.
Rats were fed either a fat-free diet supplemented with 10% menhaden oil or a control diet for four months. Intestinal brush border membranes were isolated; phospholipid fatty acid analysis revealed that the membranes from the fish-oil fed animals had higher levels of palmitoleic (C16:1) and eicosapentaenoic (C20:5) acids and lesser levels of stearic (C18:0) linoleic (C18:2) acids compared with controls. The membranes from the fish-oil fed animals had increased levels of alkaline phosphatase activity compared with controls but disaccharidase levels were equivalent in the two groups. Rocket immunoelectrophoresis studies revealed that the increase in alkaline phosphatase activity was due to an increase in the specific activity of the enzyme rather than an increase in the amount of enzyme. Membrane fluidity was assessed by fluorescence anisotropy using diphenylhexatriene and 12-anthroyl stearate as fluorescent probes. The anisotropy of both probes was similar in the two membranes. These studies indicate that fish-oil supplementation alters the fatty acid composition of the intestinal brush border membrane and increases alkaline phosphatase activity without affecting membrane fluidity. Thus the effects of changes in membrane lipid composition on alkaline phosphatase activity appear to result from changes in the local lipid environment of the enzyme rather than from changes in the biophysical characteristics of the membrane.
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