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11.
Nakazawa Miki; Kakihana Junko; Hisabori Toru; Manabe Katsushi 《Plant & cell physiology》1990,31(6):789-796
Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the PFR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A666/A280 of PR) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. PR and PFR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.051.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofPFR was at 728730 nm and that of PR was at 666667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the PRor PFR form did not result in degradation of the molecule. Therate of dark reversion of PFR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate.
2Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990) 相似文献
12.
Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts. 总被引:3,自引:1,他引:2
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S Imai T Fujino S Nishibayashi T Manabe T Takano 《Molecular and cellular biology》1994,14(11):7182-7194
13.
Nakazawa Miki; Hayashi Hidenori; Yoshida Yuhji; Manabe Katsushi 《Plant & cell physiology》1993,34(1):83-91
Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either thePR or the PFR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the PR or the PFR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of PFR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for PR (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the PR and PFR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992) 相似文献
14.
An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels 总被引:1,自引:0,他引:1
A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera. 相似文献
15.
Takashi Manabe Tsuneo Okuyama Akiko Suzuki Akiyo Shigematsu 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1981,225(1):65-71
The changes in rat plasma protein distribution after carbon tetrachloride administration were examined using two-dimensional electrophoresis, utilizing isoelectric focusing in polyacrylamide gel in the first dimension and pore gradient polyacrylamide gel electrophoresis in the second dimension. Drastic changes in amount of protein were observed at more than 20 spot positions including those of transferrin, Gc-globulin and low-density lipoprotein. The time course of the changes was examined, and the most drastic changes were observed at 2 days after carbon tetrachloride administration. 相似文献
16.
Despite the presumed importance of desmoglein, a 160-kDa glycoprotein, in desmosome formation and its possible involvement in certain blistering skin diseases, the precise location and function of this protein have not yet been firmly established. We describe here the characterization of a new monoclonal antibody, AE23, against an extracellular epitope of desmoglein. Both the AE23 epitope and another epitope, defined by the previously characterized DG3.4 antibody, reside on a 160-kDa human epidermal desmoglein as evidenced by their identical solubility profile, their coexistence in a 130-kDa desmoglein degradative product, their coadsorption by an AE23 immunoaffinity column, and the identical changes in the two antigens' electrophoretic mobility after air oxidation and deglycosylation. The AE23 epitope is resistant to various endoglycosidases, suggesting that sugar moieties are not involved. Characterization of several proteolytic fragments of this epidermal desmoglein enabled us to map the DG3.4 epitope to a 96-kDa intracellular domain and the AE23 epitope to an extracellular domain flanked by the plasma membrane and the distal N-glycosylation site(s). However, these two epitopes do not always coexist on the same desmoglein molecule. For example, tissue surveys showed that although the DG3.4 epitope is present in the desmogleins of all epithelial cell types, the AE23 epitope is limited to normal keratinocytes. Moreover, electron microscopic localization data indicate that whereas the DG3.4 epitope is detected in the submembranous plaques of desmosomes, the AE23 epitope is present in the intercellular space of both desmosomal and nondesmosomal areas. These results raise the possibility that there exist several biochemically closely related isoforms of desmoglein, one (AE23+/DG3.4+) restricted to epidermal desmosomes, one (AE23+/DG3.4-) uniformly distributed along the keratinocyte cell surface, and another (AE23-/DG3.4+) present in desmosomes of simple epithelia and basal cells of cultured keratinocytes. The uniform distribution of at least one desmoglein-related antigen in the intercellular space of keratinocytes coupled with the realization that different isoforms of desmogleins form a subfamily of cadherins suggest that desmoglein(s) may play a more general role in keratinocyte adhesion than previously appreciated. 相似文献
17.
Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation 总被引:10,自引:0,他引:10
The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation. 相似文献
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Shevin T. Jacob Patricia B. Pavlinac Lydia Nakiyingi Patrick Banura Jared M. Baeten Karen Morgan Amalia Magaret Yuka Manabe Steven J. Reynolds W. Conrad Liles Anna Wald Moses L. Joloba Harriet Mayanja-Kizza W. Michael Scheld 《PloS one》2013,8(8)