Identification of a 9 kDa gamma-crystallin fragment in human lenses.
The degraded polypeptides (M(r) less than 14 kDa) were isolated by a preparative SDS-PAGE method from water soluble (WS) and water insoluble (WI) proteins of human lenses from donors of ages between 5 and 75 years. SDS-PAGE analysis showed the presence of a major 9 kDa polypeptide species that showed an age-related increase in levels in WS-polypeptide preparations. In order to identify the parent crystallin of the 9 kDa polypeptide, the immunoreactivities of the WS- and WI-degraded polypeptides to immuno-affinity-purified anti-human alpha-, beta- and gamma-crystallin antibodies were determined by the Western blot method. The WS- and WI-9 kDa polypeptides showed immunoreactivity to only the anti-gamma-crystallin antibody suggesting it to be a fragment of gamma-crystallin. A 9 kDa species was purified by Sephadex G-50 chromatography from the WS-protein fraction of lenses from 20-30-year-old donors. The purified polypeptide showed a single protein band during SDS-PAGE and also an apparent single spot on two-dimensional gel-electrophoresis (IEF followed by SDS-PAGE). The purified preparation also showed a single major peak during reverse phase HPLC chromatography. The purified 9 kDa polypeptide showed immunoreactivity to only the anti-gamma-crystallin antibody. A polyclonal antibody raised against the purified 9 kDa polypeptide showed immunoreactivity only to a 20 kDa gamma-crystallin species. The partial N-terminal sequence analysis of the 9 kDa polypeptide showed it to be a fragment of gamma D-crystallin. Together these results show that a 9 kDa gamma D-crystallin fragment exists in increasing quantities in human lenses during aging.
Srivastava
O P
OP
Missouri Lions Eye Research Foundation, Columbia.
McEntire
J E
JE
Srivastava
K
K
D000293:Adolescent; D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D002850:Chromatography, Gel; D003459:Crystallins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis
D003459:Crystallins; D010455:Peptides