J. Alcalá

Protein composition of bovine lens cortical fiber cell membranes

Abstract A plasma membrane-enriched fraction was obtained after treatment of the water-insoluble residue of the adult bovine lens cortex with 8 m -urea for the solubilization of albuminoid (i.e. intracellular matrix). Morphological characterization of the urea-insoluble plasma membrane-rich fraction by electron microscopy showed it to consist of membrane fragments in the form of clear vesicles of various sizes. No obvious damage to the integrity of the membranes could be detected as a result of this treatment. Electrophoretic analysis of the urea-insoluble plasma membrane-rich fraction in 5·13% polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS) showed it to consist of seven major polypeptide components of estimated molecular weights (I) 200 000, (III) 68 000, (VI) 43 000, (VIII) 35 000, (IX) 27 500, (X) 22 500, and (XI) 20 000 daltons. Component III (mol. wt. 68 000) was the major PAS-positive component and probably represents the major glycoprotein fraction of the lens membranes. Components VIII, IX, X, and XI had mobilities identical to the mobilities of the soluble crystallins in the 1% SDS electrophoresis system. The predominant polypeptide component of the membrane-rich fraction, component IX (estimated mol. wt. 27 500), comprised nearly half of the protein recovered from this fraction as determined by densitometric scanning of the gels. Electrophoretic comparison with the soluble lens fraction revealed that this component had a mobility identical to that of the predominant polypeptide chain (B P ) of beta-crystallin in the 1% SDS electrophoresis system. The next most abundant component of the membrane-rich fraction, band X, comprised 24% of the protein recovered from this fraction and had a mobility identical to that of alpha-crystallin in the 1% SDS electrophoresis system. Whether the polypeptide components of bands VI–XI in the membrane-rich fraction represent homomonomers (crystallins or membrane components) or heteromonomers (crystallins and membrane components) of similar molecular size (i.e. mobility) remains to be determined.

Human lens fiber cell plasma membranes. I. Isolation, polypeptide composition and changes associated with ageing

Abstract A detailed methodology is offered for the bulk isolation of human lens fiber cell plasma membranes. The human lens fiber plasma membrane fraction is isolable as the water-insoluble and urea-insoluble but detergent-soluble material, under reducing conditions. Ultrastructural characterization confirmed the homogeneity of this fraction. Biochemical, immunological and electrophoretic analyses were conducted upon the isolated fiber plasma membranes. Analyses were conducted of whole-lens, cortical and nuclear fiber plasma membranes from fetal, newborn, 30–50, 50–60, and 60–80-year-old normal human lenses. The fiber plasma membrane fraction remained a relatively constant 0·9% of the fresh (wet) weight of the human lens throughout lifespan; the protein-lipid ratio was determined as 1:1·2 with a slight increase in the lipid factorial of older lenses (50 years and older). Thirteen (13) polypeptides, ranging in molecular weight from 12–235 kilodaltons were resolved electrophoretically for the membranes. The phospholipid-containing 25 and 27 kilodalton polypeptides were found to constitute the main intrinsic protein of the human lens fiber membranes. α-Crystallin polypeptides (20 and 22·5 kilodaltons) were consistently recovered from the membranes, and their presence confirmed immunologically only following total detergent solubilization of the membranes. Reactions of an antiserum to the main intrinsic protein of chick lens fiber membranes and the human lens membranes were negative. The major age-dependent change in the protein composition of whole-lens human fiber membranes consisted of a gradual reversal in the preponderance of the 27 kilodalton polypeptide prenatally (and at birth) by the 25 kilodalton polypeptide as the main intrinsic polypeptide of the membranes postnatally. Additional changes comprised a gradual increase in the weight fraction of the 12 kilodalton polypeptide in the whole-lens fiber membranes throughout lifespan, and a gradual decrease of the α-crystallin content of cortical fiber membranes of lenses 50 years and older.

Biochemical and structural features of chick lens gap junctions

Deoxycholate treatment of isolated chick lens fiber cell plasma membranes yields a fraction rich in gap junctions. Analysis by SDS-PAGE revealed the preponderance of a 26k dalton (MP 26k) component and a minor 43k dalton (MP 43k) component in the membranes with further specific enrichment of the MP 26k component in the gap junction rich fraction. Analysis of the isolated chick lens gap junctions by freeze-fracture and negative staining revealed aggregates of 9·0 nm intramembrane particles (connexons) arranged in a non-crystalline pleomorphic pattern. The results imply that MP 26k is the principal component of lens fiber cell gap junction connexons.

The Protein Structure of Chick Lens Fiber Cell Membranes and Intracellular Matrix

Electrophoretic analysis of the water soluble fraction of chick lens fiber cells, the urea-soluble fraction, and the urea-insoluble plasma membrane fraction was performed in 5.13% polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). Five polypeptides were identified for the water soluble fraction. One polypeptide of molecular weight 22,500 daltons corresponded to subunits of α-crystallin, three polypeptides of molecular weights 25,000 daltons, 27,500 daltons, and 37,000 daltons corresponded to subunits of β-crystallins, and one polypeptide of 43,000 daltons corresponded to subunits of δ-crystallin. The water insoluble fraction contained twelve additional polypeptides with molecular weights ranging from 41,000 to 200,000 daltons. The 8 M urea soluble fraction (albuminoid) contained the 5 crystallin polypeptides as well as 8 additional bands. The major component of albuminoid consisted of a polypeptide of 41,000 daltons not found in the water soluble fraction of the lens. The urea-insoluble fraction (cell membranes) consisted of only 8 bands, one of which corresponded in molecular size with subunits of δ-crystallin and 2 with subunits of β-crystallin. However, the presence of lipid in the major membrane component (54.0%; with a mobility of a β-crystallin subunit) suggests that this component ist not a β-crystallin polypeptide.

The presence of delta-crystallin in the plasma membrane of chick lens fiber cells

Abstract Analyses by SDS-polyacrylamide gel electrophoresis of plasma membranes isolated from a rebion of the chick lens (outer cortex) where little or no (

Regional Differences in the Polypeptide Composition of Chick Lens Intracellular Matrix

Analysis of the chick lens intracellular matrix by SDS polyacrylamide gel electrophoresis revealed that the cortical urea-soluble protein is characterized by a high content of non-crystallin polypepti

Ontogeny of chick lens β crystallin polypeptides by immunofluorescence

An anodal β -crystallin fraction and a cathodal β -crystallin fraction of adult chicken lens were injected into rabbits for the production of antisera. Immunochemic

The Structure of Chick Lens Water-Insoluble Material

The structure of chick lens water-insoluble material was analysed by electron microscopy before and after treatment with 8 m urea. It was concluded that the water-insoluble material consists of cell m

Effect of Urea on Chick Lens Proteins

The concentrations of urea commonly used to dissociate proteins caused an irreversible time-dependent loss of antigenicity in chick lens δ-crystallin, and an irreversible decrease in the electrophoret