Characterization of partially purified from porcine lens |
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Authors: | Parimal C. Sen Douglas R. Pfeiffer |
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Affiliation: | Hormel Institute, University of Minnesota, Austin, MN 55912 U.S.A. |
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Abstract: | The partial purification of from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of which can be phosphorylated by reaction with [γ-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the for and inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit from other tissues such as oligomycin, Ca2+, vanadate, , (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the ()-ATPase and activities. The K+ congeners were relatively more effective in supporting compared to activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of activity, activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed. |
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Keywords: | Protein purification Membrane fluidity Cataract (Porcine lens) DPH 1,6-diphenyl-1,3,5-hexatriene DTNB 5,5′-dithiobis-(2-nitrobenzoic acid) PCMBS SDS sodium dodecyl sulfate |
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