| Research Abstract |
Neumerous number of NTP-binding proteins were detected in the microsomal fraction of mycelia of Neurospora crassa. By the Western blotting we detected four species of proteins with molecular masses, 21 kDa, 24 kDa, 26 kDa, and 27 kDa, which were cross-reactive with the Ras-specific antibody. Among them 26 kDa protein was partially purified. The 26 kDa protein had the capacity to bind [alpha^<-32>P]ATP,[alpha^<-32>P]GTP,[alpha^<-32>P]CTP and/or [alpha^<-32>P]UTP at 1.6 x 10^<-7>M and was autophosphorylated from 1.7 x 10^<-7> M [gamma^<-32>P]ATP or [gamma^<-32>P]GTP. Using v-Ha-ras DNA as a probe, ras related genes, NC-ras 2A and NC-ras2B were cloned. cDNA of NC-ras 2A also cloned and sequenced.NC-ras 2B was closely related to NC-ras 2A, but the restriction map of the two genes were a little different. Part of the nucleotide sequence of NC-ras 2B was determined and small differences could be detected between the two genes. NC-ras 2A contained most of the consensus sequences related to ra
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s-genes in animals.
We detected 58 KDa, 77 KDa, 83 KDa and 129 KDa proteins, which were stimulated to bind [alpha^<-32>P]ATP and/or [alpha^<-32>P]GTP by the irradiation of the reaction mixture by UV-A (355 nm) for 1 sec. pretreatment of the microsomal fraction by an antibody specific to the C-terminus of transducin resulted in blocking the UV-A light stimulated [alpha^<-32>P]GTP binding to 83 kDa protein. 83 kDa protein showed also cross reactivity with the antibody showing specific staining in Western blotting. By use of C-termini of mRNAs of transducin-1 and -2, we cloned 5 independent clones. Among them one of the clone showed strong homology to NC-ras 2A. The sequencing of the gene is now under progress.
Further we detected 15 kDa protein, which was stimulated to be phosphorylated at 0ﾟC for 5 sec from [gamma^<-32>P]ATP at 4.2 x 10^<-8> M by 1 sec irradiation of blue light (465 nm). The microsomal membranes prepared from wc-1 and wc-2 showed no activity to respond to blue light in the phosphorylation of 15 kDa protein. Mixing of the membranes from wc-1 and wc-2 restored the capacity to respond to blue light in the phosphorylation of 15 kDa protein. Inhibitor of ion mobilizing ATPases, DCCD, DES and oligomycin inhibited blue light induced phosphorylation of 15 kDa protein. Blue light induced phosphorylation could slightly be detected even in 70 kDa protein. Coupled phosphorylation of 15 kDa and 70 kDa proteins were suggested. The phosphate group of 70 kDa was resistant to alkaline condition and suggested to be the phosphorylation of histidine residue. This relation was very similar to that of E. coli chemotaxis system, which include histidine phosphorylated cha A (73 kDa) and che Y (15 kDa). Ion translocating ATPase including F_0F_1-ATPase contain 15 kDa protein. To analyze the evolution of the system of light signal transmission, molecular cloning of the subunit of ATPases, NTP-binding proteins and rapidly phosphorylated 15 kDa and 70 kDa proteins may be the first step to develop the research. Less
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