FLAGELLAR MOVEMENT AND ADENOSINE TRIPHOSPHATASE ACTIVITY IN SEA URCHIN SPERM EXTRACTED WITH TRITON X-100

TLDR

Triton X‑100 extraction removes the flagellar membrane from sea urchin sperm while preserving the motile apparatus, enabling nearly all sperm to regain motility when reactivated with ATP. ATPase activity of reactivated sperm was quantified with a pH‑stat while oligomycin inhibited residual mitochondrial ATPase. Reactivated sperm beat at 32 Hz and travel 2.4 µm/beat—lower than live sperm (46 Hz, 3.9 µm/beat)—and show 0.16 µmol P_i /(min × mg protein) ATPase activity, of which ~72 % is coupled to motility; this coupled activity depends on ATP, Mg²⁺, and is suppressed by increased viscosity, altered pH or salt, with K_m values of 0.15 mM for ATPase and 0.2 mM for beat frequency.

Abstract

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25 degrees C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 microm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 microm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 micromole P(i)/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 micromole P(i)/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg(++), although some motility was also obtained with Mn(++) and Ca(++). The coupled ATPase activity had a Michaelis constant (K(m)) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "K(m)" of 0.2 mM.