An alkali-insoluble core material was isolated from the fruit bodies of Lentinus edodes after exhaustive extraction with 24% NaOH at 5 degrees C. This material consists mainly of glucan which is closely associated with chitin. Methylation analysis has shown that the glucan part of the core material (skeletal glucan) has a highly branched structure with 1,6 and 1,3 linkages in a molar ratio of 2 : 1. Stepwise enzymatic hydrolysis with basidiomycete sp. QM 806 beta-1,3-glucanase has indicated the heterogeneity of the skeletal glucan. The outer part of the skeletal glucan seems to be composed mainly of beta-1,3 and beta-1,6 glucoside linkages and has a close structural similarity to lentinan, a water-soluble beta-glucan from L. edodes. The middle part of the skeletal glucan appears to be composed mainly of beta-1,6 glucoside linkages. The innermost part of the skeletal glucan is a highly branched glucan with beta-1,6 and beta-1,3 linkages. Probably, it is associated with chitin and a small amount of amino acid polymer.

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To ascertain whether ouabain binding to human alpha1-subunit influences coexpression of rat alpha1-subunit, the ouabain-sensitive profiles of Na+,K+-ATPase activity and 86Rb+ uptake activity and ouabain binding capacity were measured in HeLa cells stably expressing rat alpha1-subunit. The ouabain-sensitive profile of ATPase and 86Rb+ uptake activity seemed to be the sum of two components, one with high and one with low apparent affinity to ouabain, which were similar to that observed in HeLa and NRK-52E cells derived from human and rat, respectively. The ATPase activity with low sensitivity to ouabain increased in simple proportion to the amount of the rat alpha1 mRNA derived from transfected cDNA, which was determined by the reverse transcription-polymerase chain reaction method. The turnover number of the human Na+,K+-ATPase activity obtained from the ratio of the Na+,K+-ATPase activity to the ouabain binding capacity is about 150/sec. The expression of the rat alpha1-subunit had no effect on the turnover numbers of the Na+,K+-ATPase activity with high affinity to ouabain estimated from the ouabain binding capacity as the active site concentration. These results suggested that the ouabain bound to human alpha1-subunit did not inhibit the ATPase activity of the coexpressing rat alpha1 in these cells.

A mutant of Saccharomyces cerevisiae defective in the cell wall beta-glucan structure was obtained. The mutant cells are extremely sensitive to (beta 1-3)-glucanase digestion and mild alkali treatment. Structural analysis revealed that the alkali-insoluble, skeletal glucan from wild type cells contains two components, a (beta 1-3) linked glucan with a laminated structure, and a highly branched glucan containing predominantly (beta 1-6) linkages. The mutant cells lack the latter component.

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