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)
Srivastava
O P
OP
Department of Physiological Optics, School of Optometry, University of Alabama at Birmingham 35294-4390.
Srivastava
K
K
Silney
C
C
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D012995:Solubility
D000596:Amino Acids; D003459:Crystallins; D010446:Peptide Fragments