Burlacu S, Tap W D, Lumpkin E A, Hudspeth A J
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021-6399, USA.
Biophys J. 1997 Jan;72(1):263-71. doi: 10.1016/S0006-3495(97)78665-X.
Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.
毛细胞的机械电转导表现出适应性，据认为这是由于机械敏感毛束内基于肌球蛋白的分子马达调节传递到转导通道的张力而发生的。为了评估毛束中肌球蛋白同工酶的酶活性，我们检测了从牛蛙球囊分离的毛束的肌动蛋白依赖性ATP酶活性。通过薄层色谱法将32P标记的无机磷酸与未反应的[γ-32P]ATP分离，使我们能够测量低至0.1 fmol磷酸盐的释放量。为了区分肌球蛋白同工酶的Mg(2 +)-ATP酶活性与其他毛束酶的活性，我们抑制了毛束肌球蛋白与肌动蛋白的相互作用，并确定了ATP酶活性的降低。N-乙基马来酰亚胺（NEM）既没有降低生理学测量的适应性，也没有降低被认为是肌球蛋白1β的120 kDa蛋白的核苷酸水解活性。肌球蛋白的NEM不敏感、肌动蛋白激活的ATP酶活性从1 mM EGTA中的1.0 fmol x s-1增加到10 microM Ca2+中的2.3 fmol x s-1。这种活性在很大程度上受到钙调蛋白拮抗剂的抑制，但不受外源钙调蛋白添加的影响。这些结果表明毛束含有具有酶活性的、对Ca(2 +)敏感的肌球蛋白分子，这与Ca2+在适应性中的作用以及肌球蛋白构成毛细胞适应性马达的假设一致。
