Yuichi Kanaoka
Hokkaido University
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Featured researches published by Yuichi Kanaoka.
Analytical Biochemistry | 1977
Keiichi Yamamoto; Takamitsu Sekine; Yuichi Kanaoka
Abstract A new fluorescent thiol reagent, N -(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), had a high molar extinction coefficient and a high quantum yield when reacted with protein SH groups. We could see directly the position of the labeled proteins in the polyacrylamide gel under ultraviolet illumination, since the maximum emission wavelength of DACM was about 480 nm. In addition, the quantum yield of DACM attached to the denatured proteins was almost identical regardless of the protein species. The properties enabled us to measure by fluorometry the amount of DACM, on a picomole level, incorporated in each protein band separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).
Biochimica et Biophysica Acta | 1970
Yuichi Kanaoka; Minoru Machida; Kimiyo Ando; Takamitsu Sekine
Abstract 1. 1.|A sulfhydryl reagent N-(p-(2- benzimidazolyl)phenyl ) maleimide (BIPM) was synthesized and the fluorescence characteristics of its derivatives were studied. 2. 2.|The fluorescence behavior agrees with an empirical rule that maleimide derivatives are practically nonfluorescent, while their addition products with sulfhydryl compounds are fluorescent. 3. 3.|BIPM may be employed as a tracer of SH-containing peptides in structural studies of proteins. Further possible applications are discussed.
Biochimica et Biophysica Acta | 1973
Yuichi Kanaoka; Minoru Machida; Mikiko Machida; Takamitsu Sekine
N-(1-Anilinonaphthyl-4)maleimide (ANM) (m.p. 207-208.5 degrees C), nonfluorescent, reacts selectively with thiols to give addition products which are strongly fluorescent (excitation maximum 355 nm, emission maximum 448 nm, in ethanol). 0.7 mole of ANM was introduced into thiol groups of egg albumin without modifying any other amino acid residues. In the fluorescence spectra of the reaction products of ANM with thiols, the quantum yields increase and the emission maxima shift towards the blue as the solvent polarity decreases. This remarkable solvent dependence was described in terms ofZ value of the solvents. ANM is thus expected to be a useful fluorescent hydrophobic probe directed to thiol groups in protein.
Analytical Biochemistry | 1972
Takamitsu Sekine; Kimiyo Ando; Minoru Machida; Yuichi Kanaoka
Abstract 1. 1. N-(p-(2-Benzimidazolyl)phenyl)maleimide (BIPM) has been used for the quantitative determination of thiol compounds. The method is based on the particular fluorescence characteristics of this reagent: its adducts to thiol compounds have strong fluorescence (λmax, ex 315 mμ, λmax, em 365 mμ), while the reagent itself has no appreciable fluorescence. 2. 2. The addition reaction of 2 × 10−6M BIPM to thiols is completed within 30 min at pH 6.85 and 0°C. A linear relation was observed up to 10−5M of the reagent between concentration of adduct and intensity of fluorescence. 3. 3. A very sensitive fluorometric determination of thiol compounds has been developed based on the above results. The detection limit was 10−6M concentration of thiols, with 0.1 ml of sample, corresponding to 0.03 μg reduced glutathione, with an accuracy of 3%. 4. 4. Using the fluorometric method, the oxidation rate of glutathione catalyzed by metal ions was examined and the contents of thiols in human lacrimal fluid and seurm were determined.
Tetrahedron Letters | 1984
Minoru Machida; Kazuaki Oda; Yuichi Kanaoka
Abstract On irradiation arylcarbothioamides (1) undergo, in the absence of oxygen, the Paterno-Buchi reaction with olefins followed by cleavage, whereas under aerobic conditions the photoreaction proceeds to give 4 and 5 probably via nitrile sulfide intermediates.
Biochemical and Biophysical Research Communications | 1992
Hitoshi Nakayama; Yasumaru Hatanaka; Eiichi Yoshida; Kozo Oka; Mayumi Takanohashi; Yuuka Amano; Yuichi Kanaoka
Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis.
Biochimica et Biophysica Acta | 1974
Takao Ohyashiki; Takamitsu Sekine; Yuichi Kanaoka
Abstract F-actin labeled with N-(1-anilinonaphthyl-4) maleimide, an SH-directed fluorescent probe, was used to analyse the transmission of the effect of Ca2+ from troponin to F-actin. 1. 1.|The fluorescence emission maximum wavenumber of anilinonaphthyl-maleimide-labeled F-actin (2.29 · 104 · cm−1-2.27·104 cm−1) indicated that hydrophobicity of the microenvironments around the thiol(s) bound with the dye is 72–76 in terms of the Z value. The emission maximum did not change by combination of the actin with tropomyosin or tropomyosin-troponin complex in the presence and absence of Ca2+, but the quantum yield and fluorescence lifetime significantly changed. The lifetime of the labeled F-actin-tropomyosin complex was restored to the level for F-actin-tropomyosin complex by adding 10−5 M CaCl2, but recovery of the quantum yield was incomplete. 2. 2.|The apparent Ca2+-binding constant obtained from the change in fluorescence lifetime, 1.3·106 M, was in good agreement with the reported values obtained from different methods. 3. 3.|In isothermal experiments, no viscosity dependence of the fluorescence polarization, P, was detected for F-actin and all its complexes. 4. 4.|Thermal dependence of P revealed that a thermally activated portion involving the fluorescently labeled thiol(s), the rotational relaxation time of which was about 40 ns, appeared on addition of tropomyosin. A thermal transition in conformation around the labeled region was also observed on adding tropomyosin, with the transition temperature of 31 °C. 5. 5.|On addition of Ca2+ to the F-actin-tropomyosin-troponin system, the thermally activated portion and the thermal transition in conformation completely disappeared. Based on the results mentioned above, the conformation changes at the labeled region are discussed in relation to the transmission of Ca2+ effect on troponin to actin.
Biochemical and Biophysical Research Communications | 1970
Hitoshi Nakayama; Kazutaka Tanizawa; Yuichi Kanaoka
Abstract Modification of the carboxyl group in trypsin by the Meerwein reagent procedure developed in our laboratories has shown that 1.6 to 1.7 carboxyl groups can be alkylated with significant loss (∼ 80%) of the BAEE activity of the enzyme. The presence of a competitive inhibitor, β-naphthamidine, strongly protects the activity. The modified enzymes retained much activity for a nonspecfic substrate. These results are interpreted in terms of preferential alkylation of the carboxyl group(s) in the binding site of trypsin with the reagent.
Tetrahedron | 1983
Masao Wada; Hideo Nakai; Yasuhiko Sato; Yasumaru Hatanaka; Yuichi Kanaoka
Abstract Based on a regioselective remote photocyclization of a pair system consisting of a phthalimide group and a dithiolanyl group, a variety of aza-cyclic compounds with methylene, ester, or amide groups in their frameworks were synthesized. The dithiolanyl group provides a removable donor, which effects a needed reaction followed by removal to give a new carbon skeleton leaving little trace of the precursor form.
Tetrahedron | 1983
Masao Wada; Hideo Nakai; Keiichi Aoe; Keishi Kotera; Yasuhiko Sato; Yasumaru Hatanaka; Yuichi Kanaoka
Abstract Based on the regioselective remote photocyclization of a pair system consisting of a phthalimide group and a terminal sulfide group in their side chain, a variety of azathiacyclic compounds containing 9- to 27-membered rings were synthesized.