Characterization of a sodium deoxycholate-activatable proteinase activity associated with betaA3/A1-crystallin of human lenses.
A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PW(XL)) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pI between 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens betaA3/A1-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal antibodies raised against betaA3/A1-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The betaA3/A1-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant betaA3/A1-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of betaA3/A1-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-betaA3/A1-C-terminal antibody. Together, the data suggest that a truncated species of betaA3/A1-crystallin exhibits proteinase activity.
Srivastava
O P
OP
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Srivastava
K
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D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D015153:Blotting, Western; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry* / Q000276:immunology; D003840:Deoxycholic Acid / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D038205:beta-Crystallin A Chain
D000906:Antibodies; C489108:CRYBA1 protein, human; C051374:Cryba1 protein, mouse; D003459:Crystallins; D011480:Protease Inhibitors; D038205:beta-Crystallin A Chain; D003840:Deoxycholic Acid; D010450:Endopeptidases