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Plant and Cell Physiology, 1985, Vol. 26, No. 8 1473-1484
© 1985

Article

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

Kazuo Ogawa and Tsuneyoshi Kuroiwa

National Institute for Basic Biology, Department of Cell Biology Okazaki 444, Japan

Most nucleases from gametes of Chlamydomonas reinhardtii need Ca2$ 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 Ca2$-EGTA [ethylene-glycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid] buffer in the reaction mixture made it possible to determine their activity at the physiological Ca2$ concentration. Nuclease C3 was not activated at low Ca2$ concentration and exhibited a sharp optimum at 10–3{small tilde}10–4 M. Nuclease C1&2 were activated at a physiological concentration of 10–6 M; increasing the Ca2$ 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.

(Received June 26, 1985; Accepted August 28, 1985)
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