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C.R. Fleschner; Rat Lens Membrane Fractional Distribution of Insoluble Calpain Lp82/85 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1237.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose. Lens specific calpains Lp82/85 are found in both the water soluble fraction (WSF) and in the water insoluble fraction (WIF). The purpose of this study was to determine the distribution of Lp82/85 among membrane fractions isolated from rat lens WIF. Methods. Rat lenses were decapsulated, homogenized and separated into the WSF and WIF by differential centrifugation. The WIF was further separated into three membrane fractions (25/45, 45/50, >=50) by discontinuous sucrose gradient centrifugation. Active calpains were detected by native casein zymography of the various lens fractions. Lp82/85 protein was detected by western blots of the fractions. Results. As expected, caseinolytic activity attributable to m-calpain, µ-calpain and Lp82/85 was found in the WSF, but only Lp82/85 activity was found in the WIF. Zymography of the membrane fractions isolated from the WIF revealed Lp82/85 activity only in the densest fraction (>=50); no activity was found in the 25/45 (the quantitatively major membrane fraction), nor in the 45/50 fractions. Western blots of the lens fractions using an Lp82/85 antibody detected Lp82/85 reactive bands (Mr=81kDa) in all fractions. The relative concentration of Lp82/85 immunoreactivity was greatest in the >=50 fraction. The concentrations of Lp82/85 reactive bands found in other lens fractions measured relative to the >=50 fraction were 0.213, 0.097, and 0.001 for the WSF, 45/50, and 25/45 fractions, respectively. An immunoreactive band for m-calpain (Mr=75kDa) was found in the WSF only. Conclusions. The lens specific calpain, Lp82/85, appears to be the only calpain which has caseinolytic activity in the WIF. Furthermore, this activity is greatly enriched in the densest fraction (>=50 fraction) isolated from the WIF. Interestingly, this fraction contains the majority of the increased urea soluble and urea insoluble protein associated with selenite-induced cataract, suggesting a possible association between membrane localization of this lens specific calpain and deposition of membrane associated, selenite-cataract-induced modified crystallins.
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