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81.
Yuriko Osakabe Kazuya Nanto Hiroko Kitamura Shinya Kawai Yuki Kondo Tomoyuki Fujii Keiji Takabe Yoshihiro Katayama Noriyuki Morohoshi 《Planta》1996,200(1):13-19
The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG
immunoglobulin G
- IPTG
isopropylthio--d-galactoside
- PAL
phenylalanine ammonia-lyase
The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008). 相似文献
82.
Purification and characterization of periplasmic alpha-amylase from Xanthomonas campestris K-11151.
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J Abe N Onitsuka T Nakano Y Shibata S Hizukuri E Entani 《Journal of bacteriology》1994,176(12):3584-3588
Xanthomonas campestris K-11151, isolated from soil, produced a periplasmic alpha-amylase of a new type. The enzyme was purified to homogeneity, as shown by several criteria. The purified enzyme showed almost the same activities on alpha-, beta-, and gamma-cyclodextrins, soluble starch, and amylose. Moreover, it was active on branched cyclodextrins, pullulan, and maltose but not on glycogen. Kinetic analysis showed that alpha-cyclodextrin was the best substrate among the cyclodextrins. The substrate specificity suggested that this enzyme had the combined activities of alpha-amylase, cyclodextrinase, and neopullulanase. 相似文献
83.
Keisuke Tsutsumi Masami Niwa Naoki Kitagawa Sei-ich Yamaga Takeo Anda †Akihiko Himeno ‡Takaya Sato Humayun Khalid Kohtaro Taniyama Shobu Shibata 《Journal of neurochemistry》1994,63(6):2240-2247
Abstract: We identified and characterized 125I-endothelin-1 (125I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (KD = 4.7 ± 1.0 × 10?10 and 1.6 ± 0.3 × 10?10M, respectively; Bmax = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ETA receptor, potently competed for 125I-ET-1 binding to sections of the microvessels with IC50 values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC50 = 5.7 ± 2.5 × 10?10 and 1.4 ± 0.2 × 10?6M for tumor microvessels, 1.8 ± 0.6 × 10?10 and 1.1 ± 0.3 × 10?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ETA subtype. 相似文献
84.
Yoshihiko Sawa Ken-ichiro Shibata Mamoru Noda Tsuguo Watanabe 《Microbiology and immunology》1994,38(4):257-262
A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG. 相似文献
85.
A physical map and clone bank of the black pine (Pinus thunbergii) chloroplast genome 总被引:1,自引:1,他引:0
T. Wakasugi J. Tsudzuki S. Ito M. Shibata M. Sugiura 《Plant Molecular Biology Reporter》1994,12(3):227-241
Black pine chloroplast DNA is 119,707 bp long. The physical map is shown and the genes are listed. Plasmid clones covering
the entire DNA sequence have been ordered and available upon request. 相似文献
86.
Phototropism of youngAdiantum fern leaves is induced by red light as well as blue light. The red light response is mediated by phytochrome. This is the
first evidence of phytochrome action in diploid fern tissue. The blue light response is mainly mediated not by phytochrome,
but probably by a blue light-absorbing pigment as in the case of almost all plants and fungi. The red light-induced phototropism
becomes detectable within 2 hr after the onset of unilateral light. The highest bending rate is about 10 degrees/hr, which
occurs between 3–5 hr after the induction of the tropic response. The bending region is about 6–8 mm from the highest point
of the coiled crozier where the growth rate becomes slow. 相似文献
87.
Kato Tomohiko; Shirano Yumiko; Iwamoto Hiroyuki; Shibata Daisuke 《Plant & cell physiology》1993,34(7):1063-1072
A lipoxygenase L-4 gene was isolated from a soybean genomiclibrary. The amino acid sequence of lipoxygenase L-4 is highlyhomologous with the partial amino acid sequence of the 94-kDavegetative storage protein, vsp94, found in paraveinal mesophyllcells in the leaves of depodded soybean plants. No L-4 expressionwas observed in maturing seeds. The L-4 gene is highly expressedin the vegetative tissues of young seedlings, including cotyledons,hypocotyls, roots and primary leaves. L-4 expression followedthe same pattern as lipoxygenase activity in cotyledons peaking3 to 5 days after germination, and returning to a basal levelby 9 days after germination. L-4 gene expression was low inthe roots, stems and leaves of 10-week-old plants. Exposureof 4-week-old plants to atmospheric methyl jasmonate increasedL-4 mRNA in leaves. Continuous pod removal from 7-week-old plantsover a 2 week period resulted in dramatic accumulation of L-4mRNA in leaves. Accumulation of the L-4 protein and three otherlipoxygenase fractions in the leaves of depodded plants wasdemonstrated by ion exchange chromatography. These results indicatethat lipoxygenase L-4 is a component of vsp94. (Received May 31, 1993; Accepted August 9, 1993) 相似文献
88.
Fragment X (LMrFX) was obtained as low molecular weight preparations from a late stage 2 plasmin digest of human fibrinogen. The thrombin-treated LMrFX preparations, which resulted in impaired polymerization, were further subfractionated into polymerized and non-polymerized components. The fractions were examined by SDS-PAGE and immunochemical methods. In polymerized fractions, more peptide bands were observed on SDS-PAGE in the reduced state than in non-polymerized fractions. Both fractions contained a similar number of internal cleavages in the A, Bβ and γ chains, which are linked by disulfide bonds. Thus, the partial deficiencies in polymerization sites of the carboxy terminal region of the γ chain and the amino terminal portions of the Bβ chain, as well as internal cleavage, were considered to participate in the impairment of the thrombin-induced polymerization of LMrFX. 相似文献
89.
Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase. 总被引:5,自引:5,他引:0
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S Shibata F Yamamoto-Goshima K Maeno T Hanaichi Y Fujita K Nakajima M Imai T Komatsu S Sugiura 《Journal of virology》1993,67(6):3264-3273
ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB. 相似文献
90.
Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia. 总被引:1,自引:0,他引:1
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Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed. 相似文献