1887

Abstract

-Glucanase activities were found associated with and their culture fluids. Mild acid treatment of the organisms led to rapid inactivation of -glucanase activities, the degree of loss increasing with the age of the cultures; the results suggested an extracytoplasmic location of the cell-associated enzymes. Most of the -glucanase activities were associated with the cell walls in organisms phenotypically resistant to amphotericin B methyl ester (AME).

Two proteins (I and II) exhibiting -glucanase activity were isolated and purified by conventional procedures from cell-free extracts, cell-wall autolysates and culture fluids of sensitive and phenotypically resistant to AME. The purified enzymes appeared homogeneous on isoelectric focusing, gel electrophoresis and ultracentrifugation, with molecular weights of 150000 (I) and 49000 (II). Both enzymes hydrolysed cell walls purified from AME-sensitive and phenotypically resistant organisms, but showed different substrate specificities and patterns of activity. Enzyme II hydrolysed (1 →3)--glucans by an endolytic mechanism releasing laminaritetraose as the initial product. Glucose was the only product released by enzyme I. The properties of the individual enzymes were unaffected by their localization or the age of the culture of the organisms.

The loosening of the polysaccharide packing by ultrasonic treatment of cell walls purified from AME-resistant organisms increased the -glucanase activities bound to the walls, but did not solubilize them. Autolysis of cell walls released 58 to 66% of their -glucanase activity in 20 h, but no further release was attained on prolonged incubation. The amount of -glucanase activity released by autolysis was increased by a variety of pretreatments. Diethyl pyrocarbonate inhibited -glucanase activity and prevented autolysis. Evidence is presented indicating that interactions with lipids, polysaccharides and other cell wall proteins may be involved in the control of the activity of the cell wall-associated -glucanases in organisms phenotypically resistant to AME.

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1982-04-01
2021-07-25
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