H. Maisel

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.

Electron microscope observations on some structural proteins of the chick lens.

Abstract Electron microscopic analysis of soluble protein extracts of chick lens fiber cells revealed two distinct morphologic components. These were chains, consisting of particles (diameter 12–15 nm) attached to a filamentous backbone (diameter 7–9 nm), and relatively straight filaments (diameter 12–14 nm). The chains and filaments were resistant to ribonuclease, but were broken up by trypsin and pronase. The chains were restricted to fiber cells and not found in lens epithelial or annular pad cells. The chains and filaments were pelleted by centrifugation at 100,000 g. Such a pellet comprised mainly alpha and beta crystallins.

A cytoskeletal protein unique to lens fiber cell differentiation

A chick lens urea-soluble polypeptide of estimated mol. wt. 49 000 daltons is unique to fiber cell differentiation and is a component of the beaded-chain filaments of the chick cytoskeleton. Antigenically related proteins are also present in the human and bovine lens. There is no similarity between this protein and actin as determined by immunological analysis and two-dimensional gel electrophoresis.

Gap junctions of chick lens fiber cells.

Abstract Gap junctions of chick lens cortical fiber cells are extensive in distribution and can be visualized by scanning electron microscopy.

The cytoskeleton of chick lens cells.

Abstract The cytoskeleton of the chick lens cells was studied by morphological and immunocytochemical methods. Actin, microtubules and intermediate (10 nm) filaments were identified in the epithelial cells. The cortical fiber cells contained intermediate filaments and dispersed chains of protein. In the nuclear fiber cells, the intermediate filaments were absent, while the protein chains were aggregated. While the size of the chain backbone corresponds to actin, the conclusive demonstration that it is actin remains to be shown.

Changes in chick lens proteins with aging.

The distribution of proteins in the whole and different parts of the chick lens was studied by immunological and electrophoretic methods. In the lenses of 15-day-old embryos, α-, β- and δ-crystallins were found in the annular pad cells and cortical fiber cells. The nuclear fiber cells contained principally δ-crystallin. In 10-day-old animals only α- and β- crystallins were found in the annular pad cells, while all proteins were present in the fiber cells. In adult animal (1–2 years old) δ-crystallin was no longer present in the cortical fiber cells. Thus, with increased age, there is a disappearance of δ-crystallin, affecting first the short fiber cells of the equatorial region and then the superficial cortical fiber cells. α- Crystallin undergoes slight changes during development. However, in the annular pad cells and cortical fiber cells, it accumulates in relatively high concentration and exhibits differences in mobility. Protein ontogeny during postnatal development is also characterized by a progressive increase of anionic β-crystallins. These last fractions seem to be a typical feature of the highly differentiated lens fibers. It may be suggested that cationic β-crystallins are synthesized mainly in the young lens fibers.

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.

Band 3 and ankyrin homologues are present in eye lens: Evidence for all major erythrocyte membrane components in same non-erythroid cell

Although immunological homologues of erythrocyte membrane proteins have been individually discovered in a wide variety of tissues and cultured cells, the major structural components of the membrane have not yet been demonstrated simultaneously in the same cell type. Thus, considerable uncertainty continues to exist concerning whether the red cell homologues form elements of a structure which is similar to or unique from the framework which supports the erythrocyte membrane. Because the red cell cytoskeletal proteins, spectrin, actin and band 4.1, have been previously found in the superficial cortex of the lens, we decided to determine whether the corresponding membrane anchoring components of band 3 and ankyrin also occur in this cell type. Using antiserum specific for band 3 and ankyrin, we report the existence of immunologically cross-reactive proteins of similar molecular weight. Because these anchoring proteins appear and disappear coordinately with the aforementioned cytoskeletal proteins during the intermediate stages of lens cell maturation, it is conceivable that an erythrocyte-like membrane structural organization may occur transiently in the eye lens.

Identification of spectrin and protein 4.1-like proteins in mammalian lens

Human, bovine, canine, and rabbit lenses were found to contain proteins which cross-react with anti-4.1 serum and which have molecular weights similar to erythrocyte proteins 4.1a and 4.1b (approximately 80 kd). Additionally, bovine, canine, and rabbit lenses contain a 4.1-like protein of approximately 125 kd which is absent from human lens. Proteins which cross-react with antibody to human erythrocyte spectrin were also detected. The human lens showed weak cross-reaction of bands of 240 kd and 225 kd, and a more intense cross-reaction of a band of 235 kd. Canine and bovine lenses showed weak cross-reaction with only the bands at 240 kd and 235 kd. The lens 240 kd band of all species also demonstrated calcium-dependent binding of calmodulin. Our results indicate that proteins related to, but distinct from, erythrocyte protein 4.1 and spectrin are found in mammalian lens.