Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.
The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.
D001665:Binding Sites; D002850:Chromatography, Gel; D003208:Concanavalin A; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009089:Mucor / Q000201:enzymology*; D009391:Nephelometry and Turbidimetry; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides; D011485:Protein Binding; D012685:Sepharose; D013696:Temperature
D011134:Polysaccharides; D003208:Concanavalin A; D012685:Sepharose; D010447:Peptide Hydrolases