Comparison of the effects of fluoride on the calcium pumps of cardiac and fast skeletal muscle sarcoplasmic reticulum: evidence for tissue-specific qualitative difference in calcium-induced pump conformation - Hawkins, C.; Xu, A.; Narayanan, N. (Elsevier)
Comparison of the effects of fluoride (NaF, 1-10 mM) on the catalytic and ion transport functions of the Ca^2^+-ATPase in sarcoplasmic reticulum (SR) vesicles isolated from rabbit cardiac and fast-twitch skeletal muscles revealed similarities as well as striking tissue-specific differences depending on the experimental conditions employed. Short preincubation (3 min at 37^oC) of cardiac or fast muscle SR with fluoride in the absence of Ca^2^+ and ATP prior to initiating enzyme turnover by simultaneous addition of Ca^2^+ and ATP to the assay medium resulted in a strong inhibitory effect of fluoride on ATP-energized (oxalate-facilitated) Ca^2^+ uptake and Ca^2^+-ATPase activity. On the other hand, when turnover was initiated by the addition of ATP to SR preincubated with fluoride in the presence of Ca^2^+ but in the absence of ATP, fluoride caused concentration-dependent stimulation of active Ca^2^+ uptake by fast muscle SR with no appreciable change in Ca^2^+-dependent phosphoenzyme (EP) formation (from ATP) or Ca^2^+-ATPase activity but inhibition of active Ca^2^+ uptake by cardiac SR with concomitant inhibition of EP formation and Ca^2^+-ATPase activity. Exposure of cardiac or fast muscle SR to fluoride in the presence of both Ca^2^+ and ATP resulted in concentration-dependent stimulatory effect of fluoride on Ca^2^+ uptake with no change in EP formation or Ca^2^+-ATPase activity; this effect diminished substantially at saturating oxalate concentration in the assay. Assessment of the effects of deferoxamine (1 mM) and exogenous aluminum (10 μM) did not indicate a requirement for aluminum in the inhibitory or stimulatory effect of fluoride. These results suggest that (a) the Ca^2^+ and ATP-deprived (E"1/E"2) but not the Ca^2^+ plus ATP-liganded (CaE"1ATP) conformation of the SR Ca^2^+-ATPase is susceptible to inhibition by fluoride in both cardiac and fast muscle; (b) the Ca^2^+-bound conformation (CaE"1) of the SR Ca^2^+-ATPase is susceptible to inhibition in cardiac muscle but is refractory to fluoride in fast muscle; and (c) the stimulatory effect of fluoride is largely secondary to its ability to mimic the action of oxalate in intravesicular Ca^2^+ trapping when the fluoride-resistant enzyme is turning over normally. Fluoride inhibited phosphorylation of the Ca^2^+-free enzyme by P"i in cardiac and fast muscle SR indicating that fluoride sensitivity of the phosphorylation site of the SR Ca^2^+-ATPase is similar in cardiac and fast muscle. In cardiac SR, disruption of the functional interaction between Ca^2^+-ATPase and its regulatory protein phospholamban, through phosphorylation of the latter (by cAMP kinase) did not alter the fluoride sensitivity of the Ca^2^+-bound enzyme (CaE"1). These results, coupled with the refractoriness of CaE"1ATP to fluoride in cardiac and fast muscle SR, suggest that a tissue-specific difference in the accessibility (reactivity) of the nucleotide binding site to fluoride upon Ca^2^+ binding to the enzyme may account for the observed difference in fluoride sensitivity of the cardiac versus fast muscle enzyme - i.e., when the ATPase is in CaE"1 conformation, its ATP binding site is 'fluoride-reactive' in the cardiac enzyme but is 'fluoride-resistant' in the fast muscle enzyme.
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Keywords: W; V; WW-YZ; U