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61.
K. Harata M. Muraki Y. Hayashi Y. Jigami 《Protein science : a publication of the Protein Society》1992,1(11):1447-1453
The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction. 相似文献
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Shin'ichi Saito Tamotsu Inoue Ichiro Kawase Hideki Hara Yoshiro Tanio Isao Tachibana Seiji Hayashi Masatoshi Watanabe Machiko Matsunashi Tadashi Osaki Tomiya Masuno Susumu Kishimoto 《Cancer immunology, immunotherapy : CII》1991,33(3):165-170
Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of125I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of125I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy. 相似文献
66.
A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland 总被引:2,自引:0,他引:2
Y Hayashi S Miwa K Lee K Koshimura A Kamel K Hamahata M Fujiwara 《Analytical biochemistry》1988,168(1):176-183
A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland. 相似文献
67.
H Munakata N Hayashi Y Yamaguchi Y Aoyagi F Ichida M Isemura 《The International journal of biochemistry》1988,20(1):113-116
1. Human chorionic tissues were incubated with [14C]leucine and/or [3H]glucosamine, and fibronectin synthesis was examined. 2. Radio-labeled fibronectin was detected in the tissue fraction of the incubation mixture, but not in the medium fraction, indicating that fibronectin is synthesized and retained in the tissue. 3. The glycopeptides derived from 3H-labeled fibronectin showed the lectin-binding characteristics similar to those from unlabeled placenta fibronectin, but different from those of plasma fibronectin. 相似文献
68.
Polyclonal antibody elicited in a rabbit against purified cytochrome P-450cc25, which catalyzes 25-hydroxylation of vitamin D3, inhibited not only 25-hydroxylation of cholecalciferol and 1 alpha-hydroxycholecalciferol, but also 16 alpha- and 2 alpha-hydroxylation of testosterone catalyzed by the purified P-450cc25 preparation. Antibody inhibition experiments with microsomes revealed that most 16 alpha- and 2 alpha-hydroxylation of testosterone and most 25-hydroxylation of cholecalciferol by male rat liver microsomes were catalyzed by P-450cc25. In order to examine the identity of cholecalciferol 25-hydroxylase and testosterone 16 alpha-hydroxylase, monoclonal antibodies recognizing three different epitopes of P-450cc25 were prepared from hybridoma clones produced by fusion of mouse myeloma cells (P3X63Ag8U1) with the spleen cells of immunized BALB/c mouse. All of these monoclonal antibodies inhibited both 25-hydroxylation of 1 alpha-hydroxycholecalciferol and 16 alpha-hydroxylation of testosterone by purified P-450cc25. These observations suggested that immunochemically indistinguishable form(s) of cytochrome P-450 catalyzed both reactions. 相似文献
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Y Wada T Matsuo I Katakuse T Suzuki T Azuma S Tsujino S Kishimoto H Matsuda A Hayashi 《Biochimica et biophysica acta》1986,873(2):316-319
A plasma prealbumin variant with a methionine-for-valine substitution at position 30 is closely associated with familial amyloidotic polyneuropathy (FAP) type I. Secondary ion mass spectrometry of the tryptic digest of a carrier's prealbumin could easily detect an abnormal peptide containing the substitution besides the normal peptide. This is a sensitive and reliable method for the diagnosis of FAP. 相似文献