. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 178 M. SAIGUSA Susceptibility of OHSS to trypsin Two milligrams of trypsin (porcine pancreatic "trypsin 1:250," Difco Laboratories) was dissolved in 20ml of hatch water. The solution was divided into aliquots and incubated at 35°C for either 75 min or 3 h. These solu- tions were then transferred to room temperature (about 25°C). Egg clusters were placed into this solution, and OHSS activity was examined for the next 4 h. Further- more, to test whether trypsin itself causes the wrapping coat to slip off the ovigerou
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 178 M. SAIGUSA Susceptibility of OHSS to trypsin Two milligrams of trypsin (porcine pancreatic "trypsin 1:250," Difco Laboratories) was dissolved in 20ml of hatch water. The solution was divided into aliquots and incubated at 35°C for either 75 min or 3 h. These solu- tions were then transferred to room temperature (about 25°C). Egg clusters were placed into this solution, and OHSS activity was examined for the next 4 h. Further- more, to test whether trypsin itself causes the wrapping coat to slip off the ovigerous hair, egg clusters were in- cubated for 4 h at 25°C. with ml of 10 ppt SW con- taining only trypsin, and at the same concentration. Gel filtration Hatch water collected from several females was cen- trifuged at 15,000 rpm for 30 min at 5°C to remove the solid materials. The supernatant was freeze-dried and was then reconstituted in 1 ml of 10 m^/Tris-HCl buffer (pH ). This test sample, containing Blue Dextran (Phar- macia) and 1 M NaCl for calibration, was applied to a Sephacryl S-200 (Pharmacia) column (45 cm X cm ), and fractions ( ml) were collected at 10-min in- tervals. The column was eluted with the Tris-HCl buffer, and the protein in each fraction was monitored with a Beckman DU-65 spectrophotometer at 280 nm. The activity of OHSS in the fractions from gel nitration was determined by the method of Shirai (1986), as follows. A series of threefold dilutions of each active fraction was prepared, and an egg cluster was immersed in each dilu- tion and tested by gentle pulling with forceps. The re- sponse—the percentage of stripped, undamaged hairs in each solution—was then plotted against the log of the dilution (Fig. 2). The potency of a fraction was expressed as the dilution producing a half-maximal effect (ED50). But because the maximal response in this assay is about 80% and the minimum is about 10% (dashed lines in Fig. 2), the ED5(j was
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