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A.M. Minaré Filho, W.B. Castro, O.S. Pereira Júnior, M.L. V. Rodrigues, R. Erasmo, R. Vanderlei; Proteasome is Present in Aqueous Humor of Rabbits . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4878.
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Some fundamental activities that make life possible, such as antigen presentation, apoptosis, protein turnover and regulation of transcriptional factors, depend on proteolytic activity, which main component is the ATP–Dependent Proteolytic System (proteasome). This complex was also described in the extracellular environment, attached to the external side of spermatozoid’s cellular membrane.
We aimed to determine if proteasome is found both in the aqueous and vitreous humors, as well as its localization in the intra or extracellular space.
We used young and healthy New Zealand rabbit specimens, of both sex. They were anesthetized and submitted to paracentesis of the anterior chamber, with subsequent aspiration of the aqueous and vitreous humors. Following this, the samples were stored in Eppendorfs and added 10% glycerol (final concentration of each was about 2%, in order to keep proteasome activity intact) and finally inserted in ice. The samples were then analyzed by SDS PAGE (20µg protein/column) and submitted to Western blotting to link proteasome subunits and ubiquitinated conjugates. The proteasome activity was evaluated using a spectrofluorimeter method with fluorogenic substrates (Suc–Leu–Leu–Val–Tyr–AMC). A specific proteasome activity inhibitor (MG132) was used in the control samples.
We could identify alpha subunits of the proteasome and ubiquitinated conjugates. Activity was preserved and could be inhibited by MG132. We also submitted them to centrifugation and HE staining of the pellet, and none cell was found.
the proteasome is do present in "free state" in the aqueous humor and vitreous, in an active form. Despite some papers could demonstrate it in other ocular tissues (lens, retina and trabecular meshwork), in our reviews we don't see any citation of the presence of this complex in the aqueous humor, an acellular environment.
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