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21.
Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-13C]Cys. The13C signals were assigned by the15N,13C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys71-Cys101 disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met73-Val74, is more accessible to solvent than the other disulfide bond. Cys35-Cys50, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys71 and Cys101 regenerates at a much faster rate than that between Cys35 and Cys50.Nomenclature of the various states of SSI that are observed in the present study
Fully oxidized-folded
native or intact (without GuHCl or DTT)
-
half reduced-folded
(Cys71-Cys101 reduced; DTT without GuHCl)
-
inversely half reduced-folded
(Cys35-Cys50 reduced; a reoxidation intermediate from fully reduced-folded state)
-
fully reduced-unfolded
(reduced by DTT in the presence of GuHCl)
-
fully reduced-folded
(an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture) 相似文献
22.
Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described. 相似文献
23.
Tetsuhiro Sakai Toshitaka Ikehara Hiroshi Miyamoto Kenichi Kaniike 《Life sciences》1981,29(23):2429-2436
Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K+ was replaced by Rb+ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K+ or Rb+ influx was increased, whereas the active cation uptake was unaffected and cell K+ was rather decreased. The serum did not alter uptake of [3H] amino acids. Also, protein synthesis inhibited in the Rb+-substituted medium was not stimulated significantly, except that observed only when the external K+/Rb+ ratio was . From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb+-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium. 相似文献
24.
25.
Mitsuhiko Akaboshi Masato Noda Kenichi Kawai Hirotoshi Maki Keizo Kawamoto 《Origins of life and evolution of the biosphere》1979,9(3):181-186
Radical formation in9 0Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results. 相似文献
26.
Kenichi Takaya 《The Histochemical journal》1976,8(1):13-23
Synopsis When cryostat sections of endocrine tissue were examined in a dark-field microscope, a brilliant granular luminescence was revealed in the endocrine cells thought to be concerned with protein or polypeptide hormone production. The sections were prepared from fresh materials either frozen in a cryostat chamber at –25°C, in dry ice-acetone, or fixed in formalin-calcium for 24 hr. The neurosecretory substance in the hypothalamus and the posterior lobe of the pituitary showed a blue luminescence; the acidophil cells of the anterior lobe of the pituitary, orange; basophil cells, green or blue; intermediate lobe cells, no luminescence; thyroid C cells, white-blue; pancreatic A cells, blue; B cells, orange; adrenomedullary cells, greenish blue; enterochromatin cells, green; and other endocrine cells in the gastrointestinal tract, blue or orange. After tearing and spreading the pituitary and hypothalamus with a pair of needles on a glass slide, and examining the teased specimen by dark-field microscopy, various cells of different luminescent colours became apparent in the anterior lobe of the pituitary, a blue fluorescent substance in the posterior lobe, and neurosecretory cell bodies in the hypothalamus. The different colours appear to be inherent in the granules of living tissues. 相似文献
27.
Yuko?T. Sato Shun Watanabe Takahiro Kenmotsu Masatoshi Ichikawa Yuko Yoshikawa Jun Teramoto Tadayuki Imanaka Akira Ishihama Kenichi Yoshikawa 《Biophysical journal》2013,105(4):1037-1044
The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30–60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation. 相似文献
28.
29.
Kenichi Tanaka Masaaki Nakayama Makoto Kanno Hiroshi Kimura Kimio Watanabe Yoshihiro Tani Yuki Kusano Hodaka Suzuki Yoshimitsu Hayashi Koichi Asahi Keiji Sato Toshio Miyata Tsuyoshi Watanabe 《PloS one》2013,8(12)