title: Extraction by lithium chloride of hydroxyproline-rich glycoproteins from intact cells of Chlamydomonas reinhardii.
abstract: A procedure has been developed to isolate and analyse the cell-wall glycoproteins of Chlamydomonas reinhardii. Under appropriate conditions, cell-wall glycoproteins can be quantitatively extracted from intact cells by aqueous LiCl. Although proteins and glycoproteins, which are presumably not related to the cell wall, are coextracted with the cell-wall subunits, these components can be readily identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as demonstrated by comparative analysis of LiCl-extracts from wild-type cells and the cell-wall-deficient mutant CW-15. Apart from the high-molecular-weight cell-wall components, two glycoproteins with apparent molecular weights (Mrs) of 36000 and 66000 were found to be present in LiCl-extracts of wild-type cells but absent in LiCl-extracts from the cell-wall-less mutant. Pulse-labeling experiments with [(3)H]proline and [(35)S]methionine revealed that the LiCl-extracts contained - in addition to the well-known cell-wall subunits - proteins of lower molecular weight, which are also preferentially labeled with [(3)H]proline. Protein components with Mrs of 68000, 44000, 36000, 26000 and 22000 were found to be more strongly labeled with [(3)H]proline than with [(35)S]methionine, whereas protein components with Mrs of 57000 and 52000 were more prominent after labeling with [(35)S]methionine. The portion of cell-wall subunits within the total amount of proteins extracted by LiCl was calculated to be at least 10% on the basis of the amount of hydroxyproline. Self-assembly of cell walls could be demonstrated after dialysis against water of a mixture of crude LiCl-extract and purified, insoluble, inner wall layers. Cell-wall glycoproteins could be enriched by gel exclusion chromatography of crude LiCl-extracts on Sepharose CL-4B columns equilibrated with 1 mol l(-1) LiCl.
authors.affiliation: Institut für Allgemeine Botanik und Botanischer Garten, Universität Hamburg, Ohnhorststrasse, 18, D-2000, Hamburg 52, Federal Republic of Germany.
authors.forename: J
authors.initials: J
authors.lastname: Voigt