Lens membranes. IV. Preparative isolation and characterization of membranes and various membrane proteins from calf lens.

A scheme is proposed for the preparative isolation of matrix-bound and matrix-free calf lens membranes, and the conditions and recoveries of the procedures have been studied. Gas chromatographic analyses have been carried out to characterize the sugars of the intrinsic glycoproteins. Galactose and N-acetyl-glucosamine are dominant sugar components and fucose, glucose and N-acetyl-galactosamine are minor constituents.Urea-treated membranes, prepared from cortex plus nucleus, contain only very small amounts of polypeptides which immunologically cross-react with α- and γ-crystallin and which can probably be classified as remnants. The weight ratio protein : cholesterol : phospholipid of these membranes amounted to 4·4 : 1 : 1·9, and the molar ratio cholesterol : phospholipid was 1·0, which is equal to those of the original lens tissue.A water-extractable protein fraction can be recovered from urea-treated membranes, which gives six bands in SDS-gel electrophoresis (mol. wt. 54 000, 47 000, 34 000, 27 000, 19 000 and 16 000). This fraction contains some α- and γ-crystallin according to immunological and electrophoretical criteria. Its major component has a molecular weight of 47 000.     

Lens membranes vi. Some characteristics of the EDTA-extractable protein (EEP) from bovine lens fiber membranes.

After urea-treatment of calf lens fiber membranes, a well defined extrinsic membrane protein fraction (EEP) can be extracted by EDTA-solution. It contains polypeptides of mol. wt. 32K and 35K (SDS-gel electrophoresis). Thin-layer gel filtration shows that without detergent no oligomers are formed. It is easily lost by its adsorption characteristics, however, it can be purified from contaminating α crystallin by gel filtration. The 35K fraction comprises components with isoelectric points at 4·6–4·7 (double band) and between 6·2 and 7·2. The 32K fraction shows components at 4·6–4·7 and 6·2 as was found by two-dimensional gel electrophoresis. The method of isolation, the isoelectric focusing pattern, and the amino acid and mol. wt. composition distinguish EEP clearly from the soluble crystallins. EEP comprises two distinct antigenic components with isoelectric points between 6·2 and 7·2. This was revealed by immunoelectrofocusing of EEP vs. anti-EEP antiserum from rabbit. Combining the results of two-dimensional electrophoresis and immunoelectrofocusing it was found that the 32K and 35K polypeptide have different antigenic properties. Traces of crystallins are difficult to remove. However, by means of specific anti-crystallin antisera it has been shown that EEP has a determinant in common with γ crystallin.