Human lens membrane proteinase: purification and age-related distributional changes in the water-soluble and insoluble protein fractions.
A proteinase from human lens membrane was purified by a procedure previously developed for a similar proteinase from bovine lenses (Srivastava, 1988a). The purification of the human proteinase was achieved by solubilization of the enzyme from membranes with 2% sodium deoxycholate followed by two consecutive passages through an Agarose A-1.5 m column. The purified proteinase exhibited molecular weight of 38 kDa on a SDS-polyacrylamide gel. A polyclonal antiserum was raised against bovine lens membrane proteinase, and used as a probe to examine distribution of the enzyme among water-soluble and insoluble proteins of human lenses of different ages. The antiserum had strict specificity to the human membrane proteinase as it showed immunoreaction to only the proteinase among human membrane proteins and crystallins. In addition, the antiserum also inhibited the proteinase activity on incubation. The Western blot of water-soluble proteins from 2-yr-old lens showed a 22 kDa immunoreactive protein, but an additional protein of 43 kD in lenses older than 19 years was observed. A similar Western blot analysis of the water-insoluble proteins from these lenses showed a single protein of 18 kDa that was identified as the subunit of the bovine lens membrane proteinase (Srivastava, 1988a). Furthermore, the immunoreactive 18 kD protein of the water-insoluble protein fractions could be solubilized with urea. The proteinase activity was found to increase with aging, as judged by the extraction of the enzyme with 2% deoxycholate from membranes of lenses of different ages and proteinase activity determination. Similarly, an age-related increase in the immunoreaction was also observed on measuring radio-iodinated protein A bound to an immunoreactive 18 kD protein of the water-insoluble protein fractions.
Srivastava
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Missouri Lions Eye Research Foundation, Columbia 65201.
Srivastava
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D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology*; D008565:Membrane Proteins / Q000302:isolation & purification; D008875:Middle Aged; D012995:Solubility
D008565:Membrane Proteins; D010450:Endopeptidases