Two-dimensional gel electrophoretic analysis of human lens proteins.
Human lens proteins from clear lenses were separated and identified using two-dimensional polyacrylamide electrophoresis. Isoelectric focusing, both equilibrium and non-equilibrium, was performed in the first dimension and SDS electrophoresis in the second dimension. Proteins were identified by Western blotting and sequencing techniques and by comparison with patterns obtained with purified crystallin fractions. Analyses were performed on total urea soluble proteins of lenses varying in age from fetal to 73 yr. Several hundred protein spots representing crystallins, cytoskeletal proteins and enzymes were resolved in the fetal lens. In the older lenses there was a dramatic increase in the number of protein species in the molecular weight range of the crystallins and a reduced number of discrete protein species visible at molecular weights greater than 50,000. Conversely, a number of proteins below approximately 15 kDa were visible even in the fetal lens. The number and amount of polypeptides in this molecular weight range were increased in the older lenses. Many of these low molecular weight species could be assigned to either the alpha-, beta- or gamma-crystallin fractions. An age dependent increase in the number of acidic species of both crystallins and other proteins, such as, glyceraldehyde 3-phosphate dehydrogenase was observed as well as the loss or mobility change of gamma-crystallin. Two-dimensional gel electrophoresis provides a sensitive and practical technique for characterizing all of the proteins of the human lens.
Datiles
M B
MB
National Eye Institute, National Institutes of Health, Bethesda, MD.
Schumer
D J
DJ
Zigler
J S
JS
Russell
P
P
Anderson
L
L
Garland
D
D
D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence; D002648:Child; D003459:Crystallins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005326:Fetal Proteins / Q000032:analysis; D005333:Fetus; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D007231:Infant, Newborn; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000737:chemistry*; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012689:Sequence Homology, Nucleic Acid
D003459:Crystallins; D005326:Fetal Proteins