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Plant and Cell Physiology, 1991, Vol. 32, No. 5 653-664
© 1991

Article

Nucleoside Triphosphate(NTP)-Binding Proteins and Endogenous ADP-ribosyl Transferase in Neurospora crassa

Kohji Hasunuma

Yokohama City University, Kihara Institute for Biological Research Nakamura-cho 2-120-3, Minami-ku, Yokohama, 232 Japan

Nucleoside triphosphate(NTP)-binding proteins were detected in the crude extract of mycelia of Neurospora crassa, which was treated with 1% Lubrol PX and fractionated by gel filtration. Protein fractions showing the capacity to bind [35S]ATP{gamma}S or [35S]GTP{gamma}S were designated as AGN1 to 6. The binding of [35S]ATP{gamma}S or [35S]GTP{gamma}S was prevented in the presence of 0.1 mM ATP or GTP except that in fractions AGN1 and 2, the presence of GTP stimulated the binding of [35S] ATP{gamma}S to ATP(NTP)-binding proteins. ATP or GTP was 1 to 2 orders of magnitude more effective than CTP or UTP in preventing the binding of [35S]GTP{gamma}S in AGN1, 2 and 5. Among these fractions AGN1, 2, 5 and 6 showed activity to hydrolyze 1 nM [{gamma}–32P]ATP or [{gamma}–32P]GTP. NTP-binding proteins bound with [35S]ATP{gamma}S or [35S]GTP{gamma}S had lower apparent molecular weights than the same proteins without bound nucleotide. Proteins bound with [35S]ATP{gamma}S or [35S]GTP{gamma}S and those [32P]ADP-ribosylated by endogenous ADP-ribosyl transferase in each fraction were analyzed by SDS-PAGE. About 20 species of ATP or ATP-GTP-binding proteins were detected, several of which were ADP-ribosylated. The binding of [35S]ATP{gamma}S or [35S]GTP{gamma}S to NTP-binding proteins was confirmed by the comparison of non-boiled and boiled samples immediately before loading to SDS-PAGE. ATP, GTP, CTP or UTP at the concentration of 0.1 mM effectively removed [33S]ATP{gamma}S or [35S]GTP{gamma}S bound to NTP-binding proteins.

(Received December 10, 1990; Accepted April 18, 1991)
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