Yano Y, Miki-Noumura T
J Cell Sci. 1980 Aug;44:169-86. doi: 10.1242/jcs.44.1.169.
Using a dark-field microscope equipped with a high-efficiency TV camera including a video tape-recorder, we recorded the sliding movement between outer doublet microtubules of the demembranated axonemes of sea-urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella by adding ATP and trypsin at 25 degrees C. The time and length of the sliding doublet microtubules from axonemes were measured directly from the image on the picture monitor from the video tape. The sliding velocity was almost constant in the range from 0 to 2% polyethylene glycol concentration in the reactivation medium and decreased a little at more than 2%. We prepared various lengths of axoneme fragments by homogenizing whole axonemes and found that the shorter fragments showed similar sliding velocity to that of longer ones at less than 200 microM ATP, but slightly decreased speed at more than 500 microM. ATP. The sliding movement sometimes stopped and the percentage of sliding axonemes was lower below 2 micrograms/ml trypsin. Above 3 micrograms/ml, the process appeared to be more like disintegration than sliding movement, which may be due to excess digestion by trypsin. Sliding speed was therefore measured in a reactivation medium containing 2% polyethylene glycol with the addition of ATP and 2 micrograms/ml trypsin. The velocity increased in proportion to the increase in ATP concentration. Vmax was approximately 14 micrograms/s at 2 mM ATP. In order to compare the Km for the sliding velocity with that of the ATPase activity of the axonemes, we measured ATPase activity of axonemes prepared and assayed under conditions in which sliding movement in the axonemes could be induced. Neither the curve of ATPase activity nor the curve of sliding velocity plotted against ATP concentration obeyed Michaelis-Menten kinetics. The close relationship between ATPase activity and sliding velocity suggested that 'sliding-movement-coupled ATPase activity' may well be reflected in the axoneme ATPase reported here.
我们使用配备了高效电视摄像机（包括磁带录像机）的暗视野显微镜，在25摄氏度下，通过添加ATP和胰蛋白酶，记录了海胆（凹顶伪球海胆和光棘球海胆）精子鞭毛去膜轴丝外双联体微管之间的滑动运动。从录像带的图像监视器上直接测量轴丝双联体微管滑动的时间和长度。在再激活介质中，聚乙二醇浓度在0至2%的范围内，滑动速度几乎恒定，超过2%时略有下降。我们通过将完整轴丝匀浆制备了各种长度的轴丝片段，发现在ATP浓度低于200微摩尔时，较短片段的滑动速度与较长片段相似，但在ATP浓度超过500微摩尔时，速度略有下降。滑动运动有时会停止，在胰蛋白酶浓度低于2微克/毫升时，发生滑动的轴丝百分比更低。超过3微克/毫升时，这个过程似乎更像是解体而不是滑动运动，这可能是由于胰蛋白酶过度消化所致。因此，在含有2%聚乙二醇、添加ATP和2微克/毫升胰蛋白酶的再激活介质中测量滑动速度。速度随ATP浓度的增加成比例增加。在2毫摩尔ATP时，Vmax约为14微克/秒。为了比较滑动速度的Km与轴丝ATP酶活性的Km，我们测量了在能诱导轴丝滑动运动的条件下制备和检测的轴丝的ATP酶活性。以ATP浓度为横坐标绘制的ATP酶活性曲线和滑动速度曲线均不符合米氏动力学。ATP酶活性与滑动速度之间的密切关系表明，这里报道的轴丝ATP酶可能很好地反映了“与滑动运动偶联的ATP酶活性”。
