Abstract
Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 417-436 |
| Number of pages | 20 |
| Journal | Current Eye Research |
| Volume | 10 |
| Issue number | 5 |
| DOIs | |
| State | Published - 1991 |
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ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience
Cite this
Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton. / FitzGerald, Paul G; Graham, Dcbra.
In: Current Eye Research, Vol. 10, No. 5, 1991, p. 417-436.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Ultrastructural localization of aa-crystallin to the bovine Lens fiber cell cytoskeleton
AU - FitzGerald, Paul G
AU - Graham, Dcbra
PY - 1991
Y1 - 1991
N2 - Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.
AB - Monoclonal antibodies to the bovine aA-crystallin were developed and used to probe the relationship between aA-crystallin and the bovine lens fiber cell Plasma Membrane-Cytoskeleton Complex (PMCC). Superficial bovine lens cortex was washed by repeated homogenization/centrifugation to remove "soluble protein." The resulting Plasma Membrane-Cytoskeleton Complex was covalently immobilized to inert resin, and extensively buffer washed. SDS PAGE and immunoblot analysis of both the covalently immobilized PMCC and of the sequentially-generated subcellular fractions shows that most of the lens alpha crystallin is "soluble" and readily extracted with physiologic buffers. However, this data also shows that 1) Non-alpha crystallins are progressively and quantitatively extracted from the PMCC with buffer, 2) An irreducible level of non-covalently bound alpha crystallin is achieved which cannot be readily extracted from the PMCC, even with 2 M urea, 1% NP40 or 0.4M KC1. Electron microscope level immunocytochemistry was performed on both the covalently immobilized PMCC, as well as on buffer-extracted thick frozen sections, using monoclonal antibodies to the αA-crystallin. The results show a very heavy labelling of both intermediate filaments and beaded filaments, but little or no labelling of fiber cell membranes. The data presented argues that a subtraction of the total αA-crystallin is strongly associated with the fiber cell cytoskeleton complex, and constitutes a quantitatively major component of the lens cytoskeleton fraction.
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U2 - 10.3109/02713689109001750
DO - 10.3109/02713689109001750
M3 - Article
VL - 10
SP - 417
EP - 436
JO - Current Eye Research
JF - Current Eye Research
SN - 0271-3683
IS - 5
ER -