Covalent modification at the C-terminal end of a 9 kDa gamma D-crystallin fragment in human lenses.

The presence of a 9-kDa gamma D-crystallin fragment among water-soluble (WS) and water-insoluble (WI) proteins of human lenses was investigated using individual site specific antibodies to the N- and C-terminal regions of the molecule. The polyclonal antibodies were raised against nonapeptides corresponding to the N- and C-terminal ends and are referred to as anti-9-kDa-N and anti-9-kDa-C antibodies respectively. On Western blot analysis of WS and WI proteins from lenses of donors of different ages, the WS9-kDa species showed immunoreactivity to both the anti-9-kDa-N and anti-9-kDa-C antibodies whereas WI 9 kDa species showed immunoreactivity to only the anti-9-kDa-N antibody. This suggested that possible modification had occurred at the C-terminal region of the WI 9-kDa polypeptide. The 9-kDa species of WS, water-soluble-high-molecular-weight (WS-HMW), water-insoluble-urea-soluble (WI-US) and water-insoluble-urea-insoluble (WI-UI) protein fractions was purified by preparative SDS-PAGE separation followed by HPLC on a C-18 column. Two forms of 9-kDa species were isolated from the WS proteins; one associated with the gamma-crystallin, immunoreactive to both the antibodies, and the other associated with high-molecular-weight protein, immunoreactive to only the anti-9-kDa-N antibody. In contrast, only one form of the 9-kDa species, immunoreactive to the anti-9-kDa-N antibody could be detected in the WI-US and WI-UI protein fractions.(ABSTRACT TRUNCATED AT 250 WORDS)