|Budget Amount *help
¥7,100,000 (Direct Cost : ¥7,100,000)
Fiscal Year 1991 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1990 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1989 : ¥6,000,000 (Direct Cost : ¥6,000,000)
Procedure for starfish gonad-stimulating substance was reexamined and two series (I and II) of the procedure were established. I-1. nerve collection, I-2. GSS extraction (homogenization in acid ethanol), I-3. delipid (partition with chloroform and ether), I-4. desalting (partition chromatography with Poragel PN in PrOH step-wise elution), I-5. gel-permeation chromatography ( Sephacryl S-200), I-6. isoelectric forcusing electrophoresis, I-7. normal phase partition chromatography (Licrosorb diol gel in PrOH gradient elution), I-8. reverse phase partition chromatography (ODS gel in acetoniterile gradient elution), and II-1, 2, 3. the same as the first series, II-4. desalting and gel permiation chromatography (sephadex G-50), II-5. ion-exchange chromatography ( ES-50ZN gel), II-6. reverse phase partition chromatography (ODS gel in acetoniterile gradient elution), II-8. second reverse phase partition chromatography (ODS gel in isocratic elution: 0.1% trifluoroacetic acid-10% prOH-18% acetoniterile).
Through the first series, GSS of Asterias amurensis was isolated and partial aminoacid sequence was analyzed (AEKYVGMGSYMALVGR from the N terminal). Based on the sequence a peptide was synthesized. Although the synthesized peptide neither showed the GSS activity nor inhibitory activity for GSS, its antiserum interacted with the native GSS of both Asterias and Asterina.
DNA probes based on the aminoacid sequence of Asterias GSS were synthesized (sense and antisense). Positive DNA fragment have not been obtained yet. Above mentioned anti-synthesized peptide IgG will be used for detecting the possible GSS DNA.