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221 results on '"Hara, Kiyotaka Y."'

41. Additional file 1 of Production of transglutaminase in glutathione-producing recombinant Saccharomyces cerevisiae

Author

Hirono-Hara, Yoko, Miyuu Yui, and Hara, Kiyotaka Y.

Abstract

Additional file 1: Figure S1. Cross-linking assay using BSA as a substrate after incubation for 120 min with several volumes of SmTG solution extracted from the BY4741-TG strain. Lane 1, molecular size marker; lane 2, BSA only; lanes 3, 4 and 5, 1×, 5× and 10×SmTG (+BSA), respectively; lanes 6, 7 and 8, 1×, 5× and 10×SmTG (−BSA), respectively. Figure S2. Effect of adding reduced glutathione to polymerization of caseinate by SmTG. SDS-PAGE profiles show the protein pattern of caseinate after incubation with SmTG. 1×SmTG extracted from the BY4741-TG strain was used. Lane 1, molecular size marker; lane 2, caseinate only; lanes 3, 4, 5, 6, 7 and 8, caseinate, SmTG and 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mM glutathione, respectively; lane 9, SmTG only. The polymerized caseins (molecular weight > 100 kDa) presented in the top panel were recovered and measured as shown in Fig. 2. Figure S3. Growth and glutathione production of S. cerevisiae expressing SmTG. (a) Time course of cell growth. Solid and dotted lines represent the BY4741-TG and GCI-TG strains, respectively. (b) Glutathione concentrations in 1×SmTG solutions extracted from the BY4741-TG strain (black) and GCI-TG strain (white). (c) Expression level of SmTG after cultivation for 24 h. lane 1, BY4741-TG strain; lane 2, GCI-TG strain. Expression level of SmTG was calculated from the intensity of each SmTG band and represented as relative values (%). The values are means and the error bars show the SD (n = 3).

Published
2021
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45. Catalysis and rotation of [F.sub.1] motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40[degrees] substep rotation

Author

Shimabukuro, Katsuya, Yasuda, Ryohei, Muneyuki, Eiro, Hara, Kiyotaka Y., Kinosita, Kazuhiko Jr., and Yoshida, Masasuke

Subjects
Proteins -- Research, Science and technology
Abstract

[F.sub.1], a water-soluble portion of [F.sub.0][F.sub.1]-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central [gamma]-subunit rotates in the [alpha]3[beta]3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80[degrees] substep rotation, interim dwell, and rapid 40[degrees] substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of [F.sub.1] subcomplex ([[alpha].sub.3][[beta].sub.3][gamma]) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant [F.sub.1] and hydrolysis of a slowly hydrolyzable substrate ATP[gamma]S (adenosine 5'-[[gamma],-thio]triphosphate) by wild-type [F.sub.1]. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP[gamma]S hydrolysis by the mutant [F.sub.1] ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on [F.sub.1] occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Published
2003

47. Pause and rotation of [F.sub.1]-ATPase during catalysis

Author

Hirono-Hara, Yoko, Noji, Hiroyuki, Nishiura, Masaya, Muneyuki, Eiro, Hara, Kiyotaka Y., Yasuda, Ryohei, Kinosita, Kazuhiko, Jr., and Yoshida, Masasuke

Subjects
Catalysis -- Evaluation, Enzymes -- Evaluation, Science and technology
Abstract

[F.sub.1]-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120 [degrees] rotation of the central [gamma] subunit relative to the surrounding [[alpha].sub.3][[beta].sub.3] ring. Here, we show that the rotation of [F.sub.1]-ATPase spontaneously lapses into long ([approximately equal to] 30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of [F.sub.1]-ATPase previously known as the ADP-Mg inhibited form in which [F.sub.1]-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the [gamma] subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90 [degrees] position.

Published
2001

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