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UBC Theses and Dissertations

Studies on the role of Ca²⁺ in the pancreatic acinar cell Ansah, Twum-Ampofo

Abstract

Plasma membrane-enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density centrifugation techniques were found to contain a Ca²⁺-stimulated ATPase activity. This enzyme was Mg²⁺-dependent and was stimulated by calmodulin and thus was similar to the (Mg²⁺ + Ca²⁺)-ATPase activity observed in other tissues. The specific activity of the (Mg²⁺ + Ca²⁺)-ATPase present was 4-5 fold higher than that present in crude membrane preparations and 80- 100 fold higher than that present in homogenates. The (Mg²⁺ + Ca²⁺)-ATPase activity of both crude membrane and plasma membrane-enriched preparations of cultured fibroblasts from Cystic Fibrosis (CF) patients was significantly reduced when compared to that activity observed in age-matched controls (p < 0.01 in the crude membrane preparations; p < 0.05 in the purified plasma membrane preparations). Reciprocal plots indicated that it is the maximal activation of the enzyme (V[sub Ca²⁺]) and not the affinity of the enzyme system (K[sub diss]) for calcium that is altered in CF strains. In order to determine if this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in CF was related to a decrease in the ability of these cells to transport calcium, inside-out (10) vesicles were prepared from red cells obtained from CF patients and age-matched controls. Ca²⁺-uptake activity was found to be significantly reduced in the preparations derived from CF patients. This reduction was apparent at all time points studied (10 seconds - 120 minutes) and at all free calcium concentrations used (10-150 μm). Reciprocal plots of the data revealed that the K[sub diss] for calcium of the calcium pump, was similar in control and CF samples but that the V[sub Ca²⁺] was significantly reduced (p < 0.001) in the CF preparations. Calmodulin prepared from red cell hemolysates of controls was found to stimulate Ca²⁺-transport activity to a similar extent in both CF and control samples; it did not, though, return Ca²⁺-uptake activity in CF preparations to control levels. An alteration in calcium transport activity in CF may have a number of implications that may explain some of the manifestations of the disease.

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