Abstract
Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.
Highlights
Using exogenous ‘2bI-ubiquitin,ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lensepithelialcells and lenscortex tissue
X g supernatants, degradation was continued at thesame rate fied in many organisms [36, 50,51,52,53,54] it is not surprising to yield a 58% increase in trichloroacetic acid-soluble radio- that we have identified it in supernatants of cultured rabbit activity over the control in 30 min
Ubiquitin has been established as a necessary cofactor for initiation of cytoplasmic degradation of damaged and/or rapidly turned over proteins in reticulocytes, and in some cell and the depletion of many enzymatic activities in aged as opposed to young lens tissue [30, 56, 57]
Summary
Using exogenous ‘2bI-ubiquitin,ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lensepithelialcells and lenscortex tissue. It seemed plausible that attenuation of lenticular proteolytic capabilities or processes which are required for initiation of proteolysis might be associated with the accumulation, aggregation, and eventual precipitation of proteins which occur in the aged or cataractous lens. This idea evolved from observations of damaged protein aggregates in aged, oxidatively stressed, or protease-inhibited red blood cells [8,9,10,11]. 11 To whom correspondence should be addressed at, 711 Washington St., Boston, MA 02111
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