N. Lieska

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.

Lens actin: purification and localization.

Actin was purified from the chick lens using DEAE-52 column chromatography followed by hydroxylapatite chromatography. The antibody produced against the purified actin cross-reacted specifically with lens actin from other species in addition to smooth and skeletal muscle actin and labelled the stress bundles of cultured fibroblasts. Actin was localized, using immunological methods, primarily to the plasma membrane of the epithelial and fiber cells of the chick and human lens. Actin filaments were also identified by HMM S-1 labeling in bovine cortical fiber cells. Using this procedure, the actin filaments were found throughout the fiber cell but were mainly concentrated near the plasma membrane and in cell processes. They formed a population distinct from the beaded filaments. The initial DEAE-52 column chromatography was also useful in the initial purification of lens fiber cell intermediate filament protein and two species of beta-crystallins.

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

Characterization of the myoglobin of the lampreyPetromyzon marinus

SummaryMyoglobin has been identified in the myocardium of the lampreyPetromyzon marinus, one of the most primitive of all vertebrates. This protein was isolated by ammonium sulphate fractionation and purified by successive chromatography on Ultrogel AcA 54, DEAE-Sephadex and CM-23 cellulose. The molecule differs substantially from the monomeric hemoglobins found in the lamprey as evidenced by its elution profile on DEAE-Sephadex and the fingerprint pattern of its enzymically-produced peptides. The functional significance of this protein in Agnatha is discussed.

Electron microscopy supports a fibrous substructure for lens intermediate filaments

The substructure of intermediate filaments from bovine lens cortical fiber cells was investigated by electron microscopy. Native filaments and synthetic ones regenerated from the total cytoskeletal extract and from the three purified subunits were examined. The morphologies from these various sources were essentially identical, with the exception that filaments reconstituted from one of the purified polypeptides were much shorter, very contorted and showed strings of aggregated protein. The solid cylindrical, unbranching filaments consisted of a helical arrangement of at least two, 5 nm diameter strands. The evidence indicated that each strand was composed of two, 2 nm diameter protofilaments which were also helically constructed (right-handed) with a periodicity of 11.6 nm. Intermediate filament diameter varied widely (8-14.8 nm, average 11.3 nm) and in a direct, linear manner relative to the apparent progression (helical) angle of the strands across the filaments face. These conclusions were obtained from observations on negatively stained intact filaments and reconstituted 4.4 nm fibrils and on positively stained transverse sections of fixed and embedded filaments.