Purification of Major Isozymes of Nuclease C and Production of Active Fragments by Trypsin

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Most nucleases from gametes of Chlamydomonas reinhardlii need Ca^<2+> for full activation. They have been named nuclease C and at least six species of isozymes have been found in the female gamete (Ogawa and Kuroiwa 1985a). Nuclease C1&2 and C3 were purified from the vegetative cells of this organism. Nuclease C1&2 exhibited a sharp pH optimum at 9.5, while nuclease C3 preferred a more neutral pH at 7.0-8.5. Use of the Ca^<2+>_EGTA [ethylene-glycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid] buffer in the reaction mixture made it possible to determine their activity at the physiological Ca^<2+> concentration. Nuclease C3 was not activated at low Ca^<2+> concentration and exhibited a sharp optimum at 10^<-3>〜10^<-4>M. Nuclease C1&2 were activated at a physiological concentration of 10-6 M ; increasing the Ca^<2+> concentration did not affect the activity. Nuclease C gave active fragments upon trypsin digestion. Tryptic fragments of nuclease C1&2 and C3 had molecular weights of 21,000 (referred as C6T) and 16,000 (C4T), respectively. Upon regulating the digestion, a few fragments were identified as intermediates of nuclease C6T by the in situ nuclease assay. These tryptic fragments were similar in molecular size to the minor components of nuclease C found in the cell lysates of gametes and early zygotes. This finding suggests that a minor species of nuclease C may be produced from the major nuclease C during gametogenesis.
日本植物生理学会の論文

Purification of Major Isozymes of Nuclease C and Production of Active Fragments by Trypsin

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