SUMMARY: Different exocellular extracts were isolated by concentrating the supernatants of yeast- and mycelial-phase cultures incubated in a synthetic medium. The only difference between the extracts obtained from the two phases was the presence in those obtained from mycelial cultures of a polysaccharide-rich, high-molecular-mass component, migrating in SDS-polyacrylamide gels at a position that would correspond to proteins with molecular masses of 245-265 kDa. The electrophoretic band patterns obtained before and after concanavalin A-Sepharose 4B affinity column treatments confirmed that the 245-265 kDa band was the only one of mannoprotein nature. The extract obtained from 24 h mycelial-phase culture (EA) was selected as the exocellular antigen for this work. The dry weight of EA obtained from 1 litre of culture medium was 30 mg; it contained 53% carbohydrate (18.3% glucose and 21.7% mannose measured by gas-liquid chromatography) and 10% protein. Rabbit antisera against EA were absorbed with yeast-phase organisms and used to stain Western blots of gels loaded with EAs. These antisera clearly recognized bands in the 21, 33 and 44 kDa areas. The antiserum obtained was employed to develop a double-antibody enzyme-linked immunosorbent assay for measuring EA concentrations in a culture medium. Most of the EA was released during the exponential phase of growth.


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