In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. ...The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of 32P-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific anti-sera against P-glycoprotein suggests that P35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.
Localization of the chaperone binding site Boyle, D; Gopalakrishnan, S; Takemoto, L
Biochemical and biophysical research communications,
05/1993, Letnik:
192, Številka:
3
Journal Article
Recenzirano
The hypothesis derived from models of the multi-oligomeric chaperone complex suggests that partially denatured proteins bind in a central cavity in the aggregate. To test this hypothesis, the ...molecular chaperone, alpha crystallin, was bound to partially denatured forms of gamma crystallin, and the binding site was visualized by immunogold localization. In an alternative approach, gold particles were directly complexed with gamma crystallin, followed by binding to the alpha crystallin aggregate. In both cases, binding was localized to the central region of the aggregate, confirming for the first time that partially denatured proteins do indeed bind to a central region of the molecular chaperone aggregate.
We have previously shown that biologically uncommon d-β-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of α A-crystallin from aged human lenses. The amounts ...increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-β-aspartic acids in α A-crystallin and cataract formation, we measured the d/l ratio of β-Asp-151 of α A-crystallin from both cataractous and age-matched normal human lenses. α A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the α- and β-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of β-Asp-151 of α A-crystallin from normal lenses is not statistically significant from that of α A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.
The purpose of the current study was to localize the lens membrane protein, MIP 26, in nuclear fiber cells from different regions of aged normal and age-related cataractous human lenses. Adult, ...juvenile, fetal and embryonic nuclear regions in aged normal and age-related nuclear cataractous lenses were morphologically and biochemically characterized using the technologies of immuno-gold (5 nm) labeling, semi-thin sections (200–500 nm), serial sections, DiI staining following by photobleaching, transmission electron microscopy and spot-blot analysis. Numbers of gold particles per micron length of plasma membrane and numbers of gold particles per square micron of cytosol in the embryonic-fetal and juvenile-adult nuclear regions were quantified. Results showed that the labeling pattern of MIP 26 localized to the cytosol was unique to senescent fiber cells from age-related cataractous lenses. Numbers of gold particles per square micron of cytosol in the embryonic-fetal nucleus of age-related cataractous lenses were significantly elevated (P<0.001) above numbers from fiber cells located within the adult or juvenile nuclei of the same lens or senescent fiber cells from aged normal lenses. Some of the cytosolic labeling in cataracts was localized to lipid vesicles, while the remaining labeling was negative for the lipid specific stain DiI. Spot blot analysis demonstrated that binding of the ant-MIP 26 serum was exclusive to large molecular weight components greater than 10 kDa, and not to small molecular weight fragments of the protein. The results of the current study supply further evidence that damage to membranes occurs in senescent fiber cells during age-related nuclear cataracts, resulting in the internalization of structures containing the membrane protein MIP 26.
Gap junction structures and distribution patterns of immunoreactive connexin46 (Cx46) and connexin50 (Cx50) in normal lenses and lens regrowths of rhesus monkeys were studied using electron ...microscopy and immunofluorescence double-labeling. Lens regrowths were collected from aphakic eyes of young monkeys whose natural lenses had been surgically removed 11–34 months earlier to simulate monocular congenital cataract surgery in human infants. Approximately 90% of the lens regrowths examined was in the form of a doughnut-shaped Soemmerring's ring located behind the iris. The lens regrowth consisted of lens epithelium and lens fibers enclosed within hypertrophied capsular material. The superficial equatorial region usually contained nucleated young fibers of normal appearance. The other regions consisted of many swollen fibers. Gap junctions were readily observed between fiber cells of both normal and swollen configuration in the lens regrowth. In superficial fibers, gap junctions were not associated with cytoskeletal components. In the intermediate and the deeper cortical regions, actin filament bundles were found specifically associated with gap junctions along both of their cytoplasmic surfaces. An immunofluorescence double-labeling study showed that Cx46 and Cx50 were labeled in the same gap junctions in both superficial and deeper cortical fibers of the normal lens. In contrast, in the lens regrowth strong co-labeling of Cx46 and Cx50 was only observed in the superficial fibers. The labeling for Cx50 was very weak or absent in the deeper cortex, whereas the strong labeling for Cx46 persisted throughout the major portion of the deeper cortex. The labeling for Cx46 finally disappeared in the much deeper cortex. This study shows that (1) the same distribution pattern of actin bundle/gap junction association found in normal lenses is seen in the lens regrowth, and (2) the immunoreactive distribution of Cx46 and Cx50 differ in the lens regrowths as compared with those in the normal lenses of rhesus monieys.
One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single ...alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alpha A and alpha B are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alpha A- and alpha B-crystallin genes (alpha A/BKO).
Lenses from 129SvEvTac mice and alpha A/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies.
Equatorial and axial (sagittal) dimensions of lenses for alpha A/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alpha A/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alpha A/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alpha A/BKO mice as compared to wild mice.
These results indicated that both alpha A- and alpha B-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.
Previous studies have demonstrated that partially denatured forms of the beta and gamma crystallins preferentially bind to a central region of the alpha crystallin particle, both in vitro and in ...vivo. These experiments were designed to ascertain if binding of a partially denatured protein to alpha crystallin could result in a diminished ability of alpha crystallin to protect against further protein denaturation and aggregation. A constant amount of alpha crystallin was incubated with increasing amounts of purified gamma s crystallin and then heated at 65 degrees C for 45 min. Under these conditions, the partially denatured gamma s crystallin binds to alpha crystallin. The resulting complexes were tested for their ability to protect against heat-induced denaturation and aggregation of alcohol dehydrogenase heated at 44 degrees C. As increasing amounts of partially denatured gamma s bound to alpha crystallin, the resulting complexes possessed a decreased ability to protect against heat-induced denaturation and aggregation. These results demonstrate that binding of partially denatured forms of a purified protein to alpha crystallin results in a complex with decreased ability to protect against denaturation, suggesting a possible mechanism whereby the molecular chaperone properties of alpha crystallin may be diminished in vivo.
A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogensis ...(selenite treated rats; galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.
A method is described which leads to the production of large amounts of ascites containing antitumor antibody in small numbers of mice. The antibody was then used to identify and characterize ...tumor-associated antigens on an ultraviolet light-induced murine skin fibrosarcoma. The antibody showed specific complement-dependent cytotoxicity to the homologous tumor and to an allogeneic tumor line which displayed a glycoprotein viral determinant with a molecular weight of 70,000 on its surface. Absorption of the immune ascites with other tumor cell lines removed the cytotoxicity in relation to the presence of the glycoprotein. Isolation of the tumor cell surface components binding antibody revealed two components with molecular weights of approximately 70,000 and 60,000. The Mr 70,000 component was identified as viral gp70 by peptide mapping.
Phakomatous choristoma is a rare, congenital, ocular adnexal tumor that is presumed to be of lenticular anlage based on light and electron microscopy.
The authors performed immunohistochemistry using ...standard commercially available antibodies against vimentin, S-100 protein, and several cytokeratins on a phakomatous choristoma that was excised from the right lower eyelid of a 10-week-old white boy. In addition, a battery of antibodies against lens-specific proteins, including alpha, beta, and gamma crystallins, was used.
The tumor cells showed intense immunoreactivity for all lens-specific proteins tested. The epithelial cells of the phakomatous choristoma stained positively for S-100 protein and vimentin, the intermediate filament normally found in lens epithelial cells. Keratin markers were negative.
The results of immunohistochemistry indicate that the cells of phakomatous choristoma synthesize several types of lens-specific proteins. Complementing previous light and electron microscopic studies, these data strongly support Zimmerman's conclusion that this pediatric adnexal tumor is a choristoma of lenticular anlage.
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