Hawkins C, Xu A, Narayanan N
Department of Physiology, University of Western Ontario, London, Canada.
Biochim Biophys Acta. 1994 May 11;1191(2):231-43. doi: 10.1016/0005-2736(94)90174-0.
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 degrees C) of cardiac or fast muscle SR with fluoride in the absence of Ca2+ and ATP prior to initiating enzyme turnover by simultaneous addition of Ca2+ and ATP to the assay medium resulted in a strong inhibitory effect of fluoride on ATP-energized (oxalate-facilitated) Ca2+ 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 Ca2+ but in the absence of ATP, fluoride caused concentration-dependent stimulation of active Ca2+ 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 Ca2+ 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 Ca2+ and ATP resulted in concentration-dependent stimulatory effect of fluoride on Ca2+ 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 microM) did not indicate a requirement for aluminum in the inhibitory or stimulatory effect of fluoride. These results suggest that (a) the Ca2+ and ATP-deprived (E1/E2) but not the Ca2+ plus ATP-liganded (CaE1ATP) 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 (CaE1) 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 Ca2+ trapping when the fluoride-resistant enzyme is turning over normally. Fluoride inhibited phosphorylation of the Ca(2+)-free enzyme by Pi 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.(ABSTRACT TRUNCATED AT 400 WORDS)
比较氟化物（NaF，1 - 10 mM）对从兔心脏和快肌骨骼肌分离的肌浆网（SR）囊泡中Ca(2+)-ATP酶的催化和离子转运功能的影响，结果显示，根据所采用的实验条件，既存在相似性，也存在显著的组织特异性差异。在测定介质中同时添加Ca2+和ATP启动酶周转之前，将心脏或快肌SR在无Ca2+和ATP的情况下与氟化物进行短时间预孵育（37℃下3分钟），氟化物会对ATP驱动（草酸盐促进）的Ca2+摄取和Ca(2+)-ATP酶活性产生强烈抑制作用。另一方面，当在有Ca2+但无ATP的情况下向预孵育了氟化物的SR中添加ATP启动周转时，氟化物会引起快肌SR对活性Ca2+摄取的浓度依赖性刺激，Ca(2+)-依赖性磷酸酶（EP）形成（来自ATP）或Ca(2+)-ATP酶活性无明显变化，但会抑制心脏SR的活性Ca2+摄取，同时抑制EP形成和Ca(2+)-ATP酶活性。在有Ca2+和ATP的情况下，将心脏或快肌SR暴露于氟化物中，会导致氟化物对Ca2+摄取产生浓度依赖性刺激作用，EP形成或Ca(2+)-ATP酶活性无变化，在测定中草酸盐浓度达到饱和时，这种作用会大幅减弱。对去铁胺（1 mM）和外源铝（10 microM）作用的评估表明，氟化物的抑制或刺激作用不需要铝参与。这些结果表明：（a）SR Ca(2+)-ATP酶的Ca2+和ATP缺失（E1/E2）构象而非Ca2+加ATP结合（CaE1ATP）构象在心脏和快肌中均易受氟化物抑制；（b）SR Ca(2+)-ATP酶的Ca(2+)-结合构象（CaE1）在心脏肌肉中易受抑制，但在快肌中对氟化物不敏感；（c）当抗氟化物酶正常周转时，氟化物的刺激作用在很大程度上是由于其模拟草酸盐在囊泡内捕获Ca2+的作用。氟化物抑制心脏和快肌SR中无Ca2+酶被Pi磷酸化，表明SR Ca(2+)-ATP酶磷酸化位点的氟化物敏感性在心脏和快肌中相似。（摘要截断于400字）
