Ultrastructural localization of aA-crystallin to the bovine lens fiber cell cytoskeleton
作者:
FitzGeraldPaul G.,
GrahamDcbra,
期刊:
Current Eye Research
(Taylor Available online 1991)
卷期:
Volume 10,
issue 5
页码: 417-436
ISSN:0271-3683
年代: 1991
DOI:10.3109/02713689109001750
出版商: Taylor&Francis
数据来源: Taylor
摘要:
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 theαA-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 totalαA-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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