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.
Spatiotemporal expression pattern of an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus during early development, and its potential role in larval vertical migration
