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Subcellular localization of adenylate cyclase in canine heart ventricle

University of British Columbia

Crude microsomal fractions (resuspended in 10% sucrose) of dog heart ventricles were subjected to discontinuous sucrose density gradient centrifugation in order to obtain purified fractions that were enriched in sarcoplasmic reticulum (S.R.) and contained a relatively lower concentration of plasma membrane than the crude fraction. It was observed that the fraction with the highest oxalate- supported Ca²⁺-uptake activity contained an adenylate cyclase activity. The main objectives of this study were to refine the technique of isolation of this pure microsomal fraction and to compare the properties of its indigenous adenylate cyclase activity to that contained in a sarcolemma-enriched "washed particle" fraction (Drummond, 1978). It was found that the enzyme found in the S.R.—enriched fraction differed from that in the "washed particle" preparation in that it: a) was stimulated by 10⁻⁴M epinephrine in a calcium-dependent manner; b) was significantly more stimulated by high Mg²⁺/ATP ratios; c) was more sensitive to inhibition by free calcium ion in the presence of epinephrine. French-press treatment of the microsomes before sucrose density gradient fractionation reduced plasma membrane contamination without a concomitant decrease in cyclic AMP accumulating ability. These findings are consistent with and complement the hypothesis that cardiac cell sarcoplasmic reticulum contains an adenylate cyclase activity that is not accounted for by sarcolemmal impurities in the preparations studied and that exhibits functional characteristics different from the sarcolemmal adenylate cyclase.

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2010-04-20

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http://hdl.handle.net/2429/23928

Pharmaceutical Sciences

University of British Columbia

UBCV

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