204 FREDERICK G. E. PAUTARD 



flagella that had not moved than from those that had, all later ex- 

 tractions were made from flagella prepared from immobilized sperm. 

 The alkaline-salt soluble proteins could not be precipitated, however, 

 by lowering the ionic strength. At high concentrations, a finely di- 

 vided floe occasionally sedimented after cooling the diluted solution 

 at 2°C for several days, but more usually the solution merely became 

 opalescent. Dialysis of these alkaline-salt extracts, whether diluted or 

 not, always resulted in a floe separating after two or three days treat- 

 ment at 2°C. A small portion of these floes could be redissolved in 

 0.5M KC1 after 24 hr extraction and further precipitated by lowering 

 the ionic strength. Addition of ATP and salts at pH 7.0 to the extract 

 before lowering the ionic strength resulted in an increase in the sedi- 

 mentation rate of the floe that formed. Although the x-ray diffrac- 

 tion pattern of these preparations showed undifferentiated protein, 

 microscopic, birefringent crystals, soluble in ether and partly solu- 

 ble in acetone and benzene, were often seen, suggesting lipids. Pres- 

 ent also in these dialyzed precipitates were many flat irregular plates 

 that showed no birefringence but could be etched with ether to leave 

 a framework of insoluble material. 



It was found that dense precipitates could be formed from the 

 diluted alkaline-salt extracts of fish sperm flagella by lowering the pH 

 to around 5.0. Even when the solution was very dilute and optically 

 clear, fine precipitates were often thrown down in less than a min- 

 ute at pH 5.0. These floes, when centrifuged and taken up in neutral 

 buffers, reacted with ATP and electrolytes and formed the subjects 

 for studies of contractility. A typical preparation of gel material from 

 trout sperm flagella is as follows. 



The flagella, damp with buffered 0.03M K.C1, were extracted with 

 0.5M KC1 containing 0.01 M Na 2 CO a and 0.04M NaHC0 3 for 48 hr 

 at 2°C. The extract was separated from the insoluble residue by 

 centrif ligation at about 10,000 X g for 5 min, and after removal with 

 a fine pipet was diluted tenfold with ion-free water and cooled to 

 just above freezing point. Saturated KH 2 P0 4 was then added drop 

 by drop until at about pH 5.2 the already opalescent solution pre- 

 cipitated a granular floe. This was spun down lightly for a few sec- 

 onds and gently suspended in 0.1 M KC1 buffered to pH 7.0. A few 

 hours after preparation a number of coherent gelatinous granules, 

 varying from 0.01 mra-1 mm in diameter were present in the sedi- 



