Kenzo Ohtsuki

Direct activation of guanine nucleotide binding proteins through a high-energy phosphate-transfer by nucleoside diphosphate-kinase

An in vitro study of phosphate-transfer, from the high-energy phosphates on the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinase to GDP on various guanine nucleotide binding proteins (G1, elongation factor alpha 1, recombinant v-rasH p21 protein, transducin, Gi and Go), revealed that the GDP acts as a phosphate-acceptor, in the presence of divalent cations (Mg2+ and Ca2+). This finding suggests that via phosphate-transfer, NDP-kinase may be responsible for the direct activation of various guanine nucleotide binding proteins through phosphate-transfer by the enzyme.

The inhibitory mechanism of in vitro protein phosphorylation by a nonprotein chromophore removed from neocarzinostatin

The inhibitory effect of a nonprotein chromophore removed from neocarzinostatin on protein phosphorylation by nuclear protein kinase in vitro has been studied. Low levels of the chromophore greatly inhibited protein phosphorylation in vitro. This inhibition, however, was not selectively dependent on the indicated kinases and their different phosphate acceptors (histones and non-histone protein). In contrast, the protein component (apoprotein) of neocarzinostatin did not affect the phosphorylation even at a concentration of 400-times higher than that of the chromophore. Moreover, apoprotein suppressed the chromophore-induced inhibition of protein phosphorylation in vitro in proportion to the apoprotein concentrations. Kinetic and analytical experiments suggest that the chromophore-induced inhibition of protein phosphorylation seems to be due to the binding of the chromophore to the kinases. In addition, we found that ultraviolet irradiation as well as methanol extraction can release the chromophore from neocarzinostatin, but it exhibits no inhibitory activity of DNA synthesis in growing cells. The fact that the chromophore-induced inhibition of protein phosphorylation in vitro was not sensitive to ultraviolet irradiation, which rapidly inactivated the ability of the chromophore to induce DNA degradation in vitro, suggests that there are different actions involved in the two inhibitions induced by the chromophore which is removed from neocarzinostatin.

Biochemical characterization of a specific phosphate acceptor of nuclear cyclic AMP-independent protein kinase

The regulatory mechanism of transcription involved in the phosphorylation of a 13 kDa non-histone chromatin protein from calf thymus, which is the most effective phosphate acceptor for cyclic AMP-independent protein kinase purified from the nuclei of mouse spleen cells, by the kinase has been studied in vitro. An analytical study of the circular dichroism (CD) spectra of the 13 kDa protein under different conditions showed that it underwent a major conformational change when incubated with DNA. The presented data suggest that the DNA-induced conformational change may result in a great increase of the 13 kDa protein phosphorylation by the kinase in vitro. Mg2+ (8-10 mM) enhanced the binding of the protein to DNA. Furthermore, the phosphorylated 13 kDa protein stimulated elongation of RNA synthesis by RNA polymerase II from calf thymus. However, neither the 13 kDa protein nor the phosphorylated 13 kDa protein had any affect on DNA synthesis. The available evidence suggests that the 13 kDa protein may play a role in the regulation of transcription through its phosphorylation by the kinase in vitro.

Complete recovery of the phosphoenzyme-forming activity of nucleoside-diphosphate kinases after reconstitution of their subunits

Two distinct subunits [α‐subunit (M r 21 000, pI 7.6) and β‐subunit (M r 19000, pI 6.5)] of nucleoside‐diphosphate (NDP) kinases highly purified from HeLa S3 cells can be separated by FPLC using a Mono P column in the presence of 6 M urea and 1% pharmalyte (pH range between 5.0 and 8.0). Comparatively high [32P]‐phosphate incorporation was detected when these two subunit fractions were reconstituted in vitro. Available evidence suggests that these two enzyme subunits are necessary for the formation of phosphoenzyme, which functions as an intermediate in NDP kinase action