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Rajendra Gangalum, Joseph Horwitz, Sirus Kohan, Ishanee Dighe, Suraj Bhat; Presence of αA-crystallin and αB-crystallin as two Independent Proteins in the Developing Ocular Lens: Evidence from Transmission Electron Microscopy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4959.
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The two small heat shock proteins, αA-crystallin (αA) and αB-crystallin (αB) are expressed independently in various non-lens tissues yet they are considered to be the two subunits of one single monolithic entity, α-crystallin in the ocular lens. We have previously suggested that they may exist as two independent proteins in the lens also (Bhat et al., Eur J Biochem 202, 775-781, 1991), however, experimental evidence for their independent existence within the lens has thus far remained elusive. We have investigated, if indeed these two α-crystallins (αA and αB) are two subunits of one single protein, α-crystallin or two independent proteins within the developing ocular lens.
Biochemical characterizations were done using established sucrose density gradient fractionations. Two specific non-interfering antibodies (one against αA and the other against αB) along with other commercially available antibodies against different protein markers representing various subcellular compartments were used. TEM (Transmission Electron Microscopy) and immunogold localization in rat lens fiber cell thin sections were done with 12 nm and 18 nm Gold particles conjugated to specific secondary antibodies (double labeling).
Biochemical fractionation of post mitochondrial supernatants show that both αA as well as αB are membrane associated proteins, which do not fractionate together. In vivo analyses with TEM and immunogold labeling with anti αA and/or anti αB along with markers (such as Ribophorin-1 for Rough ER) and HSP90 (for smooth membranes) indicates that αA and αB do not localize in the same subcellular membrane compartment.
The data obtained with developing rat ocular lens fiber cells reveals that αA and αB are two independent proteins that do not colocalize to the same subcellular membrane compartment. These data suggest a revision of historically held belief that α-crystallin is a monolithic entity composed of two subunits αA and αB.
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