Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates.

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.

Protease activities in cultured beef lens epithelial cells peak and then decline upon progressive passage.

Beef lens cells in culture are readily obtained and provide many opportunities to study phenomena related to cell differentiation and maturation, environmental stress, disease, and perhaps mechanisms of transformation. Although altered rates of proteolysis are known to accompany these phenomena, the proteolytic activities available in cultured beef lens epithelial cells have not been documented. In this work are documented the specific activities, based on protein and DNA content, of neutral exo- and endopeptidase, cathepsins B- and D-like enzymes and acid phosphatase in lens epithelial cortical and core tissue and in cultured epithelial cells at passages 1-43. Maximal activity of each protease occurs almost routinely at passage 5 or 9, reaching values of approx. 1400-, 0.77-, 4520-nmol min-1 per mg protein for neutral exopeptidase (passage 5), neutral endopeptidase (passage 5) and cathepsin B (passage 5) respectively, and 7.1 micrograms trichloroacetic acid soluble peptide min-1 per mg protein for cathepsin D (passage 15). On a microgram-1 DNA basis, the maximal specific activities for the same enzymes were 48 (passage 5), 0.03 (passage 5), 283 (passage 9), and 0.5 (passage 9) respectively. In subsequent passages, the specific activities declined to values which were similar to or lower than the specific activities observed for these proteases in lens epithelial tissue.

Partial purification and characterization of an ovarian tripeptidyl peptidase: a lysosomal exopeptidase that sequentially releases collagen-related (Gly-Pro-X) triplets.

The pregnant hog ovary is a rich source of a novel exopeptidase that catalyzes the release, at pH 5.0, of collagen-related (P3-P2-P1) "triplets" such as Gly-Pro-Met, Gly-Pro-Arg, and Gly-Pro-Ala from arylamide derivatives, provided the N termini are unsubstituted. Corresponding derivatives of related (P2-P1) dipeptides (Pro-Met, Pro-Ala) or (P1) amino acids (Met, Arg, Ala) are not attacked. The enzyme was purified 58-fold from a detergent extract of a water-extracted tissue residue. Activity was determined on Gly-Pro-Met-2-naphthylamide at pH 4.5 and 37 degrees C (Km 0.45 mM; Vmax 722 nmoles/min/mg protein). The responsible Mr 55,000 exopeptidase, termed "tripeptidyl peptidase", forms high-Mr aggregates, belongs to the serine catalytic class, and has a lysosomal localization. Gly-Pro-Ala triplets were released sequentially at pH 5.0 from a Mr 14,000 polypeptide, poly(Gly-Pro-Ala-). When this reaction was coupled to that of homologous dipeptidyl peptidase II, the liberated tripeptides were reduced to dipeptides and free amino acids: (Gly-Pro-Ala)n----nGly-Pro-Ala----nGly-Pro + nAla.

Identification of a cathepsin b-like protease in the crevicular fluid of gingivitis patients.

Crevicular fluid from gingivitis patients contains significant levels of a cysteine protease which was characterized as the lysosomal protease cathepsin B, as judged by substrate specificity, thiol dependence, pH optimum, kinetic parameters, pH stability, and inhibitor sensitivities. A highly-sensitive fluorometric assay procedure was used to establish the mean level of cathepsin B activity for 25 gingivitis patients.