Calcium binding properties of purified zymogen granule membrane of pig pancreas. Evidence for calcium binding proteins.
Ca2+ binding properties of purified zymogen granule membranes of pig pancreas have been measured: Binding increased linearly with Ca2+ concentration in the medium up to the micromolar range; in the millimolar range a sharp rise in binding capacity was observed. Binding increased with pH both at low and high concentrations of Ca2+. It was insensitive to Na+ and K+ ions at concentrations up to 100 mM. Mg2+ was inhibitory in the millimolar range whereas La2+ and Tb3+ were inhibitory in the micromolar range. The Ca2+ binding components of zymogen granule membranes were identified by two methods: (1) by measuring 45Ca2+ binding after counter-ion electrophoresis and (2) by Stain's-all (forms a complex with Ca2+ binding proteins absorbing maximally at 600 nm), after SDS-polyacrylamide gel electrophoresis. The first method, counter-ion electrophoresis, indicated that most of the 45Ca2+ was associated with an acidic band which could be subsequently subfractionated by SDS-polyacrylamide gel electrophoresis in five bands: 66, 57, 30, 27 and 22.5 kDa. The second method, Stain's-all, revealed six positive polypeptides after SDS-polyacrylamide gel electrophoresis of native zymogen granule membranes' two were unreactive after neuraminidase treatment (130 and 92 kDa, respectively), whereas four other bands were still reactive (66, 57, 43, 30 kDa, respectively.) Ca2+ binding was also measured on intact zymogen granules: the binding capacity was higher than for zymogen granule membranes. Among the Ca2+ binding proteins of the zymogen granule membrane only one is apparently located on the granule external surface: the 30 kDa polypeptide. If Ca2+ directly facilitates fusion of zymogen granules with plasma membrane by a Ca2+-protein interaction, then this protein is a presumptive candidate to play such a key role.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D013552:Swine
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D008274:Magnesium; D002118:Calcium