Normal structure and function of the lung parenchyma depend upon elastic fibers. Amorphous elastin is biochemically stable in vitro, and may provide a metabolically stable structural framework for the lung parenchyma. To test the metabolic stability of elastin in the normal human lung parenchyma, we have (a) estimated the time elapsed since the synthesis of the protein through measurement of aspartic acid racemization and (b) modeled the elastin turnover through'measurement ofthe prevalence of nuclear weapons-related "C. Elastin purified by a new technique from normal lung parenchyma was hydrolyzed; then the prevalences of D-aspartate and "4C were measured by gas chromatography and accelerator-mass spectrometry, respectively. D-aspartate increased linearly with age; K,,.,p (1.76x 10-3yr-') was similar to that previously found for extraordinarily stable human tissues, indicating that the age of lung parenchymal elastin corresponded with the age of the subject. Radiocarbon prevalence data also were consistent with extraordinary metabolic stability of elastin; the calculated mean carbon residence time in elastin was 74 yr (95% confidence limits, 40-174 yr). These results indicate that airspace enlargement characteristic of "aging lung" is not associated with appreciable new synthesis of lung parenchymal elastin. The present study provides the first tissue-specific evaluation of turnover of an extracellular matrix component in humans and underscores the potential importance of elastin for maintenance of normal lung structure. Most importantly, the present work provides a foundation for strategies to directly evaluate extracellular matrix injury and repair in diseases of lung (especially pulmonary emphysema), vascular tissue, and skin. (J. Clin. Invest. 1991.
The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.
Considerable attention has been focused upon alpha-1-antitrypsin deficiency because of the insights into the pathogenesis of human pulmonary emphysema that may derive from study of deficient subjects, and because of evolving therapeutic strategies that may slow the progression of lung disease in affected persons. We have applied an automated immunoassay for alpha-1-antitrypsin to plasma samples from 20,000 blood donors. Seven PI Z antitrypsin-deficient persons were identified and confirmed; this is more than twice the number predicted from previous estimates of the Z allele frequency in the St. Louis area. Five of the subjects were further evaluated. We anticipate that this assay, if utilized to screen large populations, could identify many alpha-1-antitrypsin-deficient persons for study of the natural history of lung and liver disease associated with the deficiency. These subjects would be potential candidates for early intervention strategies to prevent the development of lung disease. The surprisingly high prevalence of deficient persons indicates that direct screening is the best method for prevalence estimation of genetic disorders.
In recent years observations at the level of individual atoms and molecules became possible by micros-copy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholip-ids carrying one rhodamine dye molecule. For this methodology , fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bio-science-for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoi-chiometries of individual components. The ultimate goal of high-sensitivity detection schemes is observation on the single molecule level. This came into reach by the invention of scanning probe microscopy (1, 2), which has since brought a wealth of new insights (3). Optical methods allowed for detection of single atoms (4). The effective light conversion in fluorescent molecules made it possible to detect single fluorophores in liquids by confocal fluorescence mi-croscopy (5-8) and to perform high-resolution spectroscopy of single dye molecules at low temperature (9-12). The first true imaging of single dye molecules by optical means was achieved by scanning near-field optical microscopy (13). This method is unique in reaching a spatial resolution of 14 nm, much below the optical diffraction limit but restricted in its application to immobile objects. Very recently, single fluorescence labeled myosin molecules on immobilized actin filaments were imaged by conventional microscopy and illumination times of seconds (14). It would be of interest for many applications, especially in bioscience, to extend microscopy to visualization of single fluorophores in motion. To our knowledge, such imaging has not been reported to date. Here we show that the motion of single dye molecules can be visualized by conventional fluo-rescence microscopy by extending the time resolution into the millisecond range. For this, we used epifluorescence micros-copy with argon-ion laser excitation and imaging onto a highly-sensitive liquid-nitrogen-cooled CCD-camera. Optical parts were carefully selected to achieve an efficiency for the detection of emitted fluorescence as high as 3%, while scattered light was blocked effectively. For demonstration of the potentials of observing individual mobile molecules we have chosen a fluorescence-labelled lipid in a fluid lipid membrane as a most appropriate system. It uniquely permitted to use results obtained at high surface densities of labelled lipid for The publication costs of this article were defrayed in part by p...
The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated ␣, , and ␥, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REG␣⌬i, REG⌬i, and REG␥⌬i. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REG␣⌬i and REG␥⌬i formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REG⌬i exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REG protein. REG⌬i was able to form hetero-oligomers with a single site, monomeric REG␣ mutant and with REG␣⌬i. At low concentrations, the REG␣⌬i/REG⌬i hetero-oligomers stimulated the proteasome less than REG␣/REG oligomers formed from wild-type subunits, and the reduced activation by REG␣⌬i/REG⌬i was due to removal of the REG insert, not the REG␣ insert. These studies demonstrate that the REG␣ and REG␥ inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REG insert reduces binding of this subunit and REG␣/REG oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.The proteasome is a major proteolytic organelle in the cytosol and nucleus of eukaryotic cells (1-3). The enzyme is a cylindrical structure containing 28 subunits arranged as four rings of seven subunits each. The two end rings are composed of catalytically inactive subunits belonging to the ␣ subunit family based on homology to proteasome subunits from the archebacterium, Thermoplasma. The two central rings are composed of members of the  subunit family (4 -6), some of which are proteolytically active (7). Crystal structures of Thermoplasma and yeast proteasomes reveal that the  subunits form a central proteolytic chamber far from the particle's surface and that entry to this central chamber is greatly restricted (8, 9).By itself, the proteasome does not degrade intact proteins. Association with a 19 S regulatory complex converts the proteasome to the 26 S protease, an energy-dependent enzyme capable of degrading intact proteins (10 -13), including those marked by ubiquitin (14). The proteasome also binds an 11 S protein complex, which we have termed REG and others have named PA28 (15-17). REG binding to the proteasome can stimulate peptide hydrolysis as much as 100-fold. Human red blood cell REG is composed of two ϳ30 KDa subunits, REG␣ and REG. These two proteins are closely related to each other and to a third protein, REG␥ (18). The three proteins show extensive sequence similarities, except for a region of 15-32 amino a...
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