PURIFICATION AND PARTIAL CHARACTERIZATION OF THE HIGH AND LOW-MOLECULAR-WEIGHT FORM (S-FORM AND F-FORM) OF INVERTASE SECRETED BY ASPERGILLUS-NIDULANS

Two forms of secreted invertase have been purified from Aspergillus ni dulans by ion-exchange and gel-filtration chromatography. S-invertase gave a single, broad, glycoprotein band on PAGE and SDS-PAGE correspon ding in size to 185 and 78 kDa, respectively, compared with 94 and 110 kDa for F-invertase. The carbohydrate of S-invertase contained mainly mannose (14%) and less galactose (5%) whereas the F-form yielded main ly galactose (29%) and less mannose (12%). Three sharp bands of enzymi cally active glycoprotein for both the S-form (PI 4.9-5.2) and the F-f orm (PI 3-4.2) were observed after isoelectric focusing. Deglycosylati on with Endo H simplified this pattern to one enzymically active prote in band (PI 5.2). The aglycoenzymes gave narrow bands on PAGE and SDS- PAGE corresponding to 115 kDa and 60 kDa respectively for both S- and F-forms. The specific activity of S-invertase was three-fold higher th an that of F-invertase both before and after deglycosylation. The K-m values of the two forms of invertase were very similar. Significant ho mology existed between the N-terminal amino-acid sequences of S-invert ase (and of internal peptides derived from it) and sequences of invert ase from other species. It is suggested that the higher carbohydrate c ontent in F-invertase results in the native enzyme existing as a monom er and having a greater negative charge and lower specific enzyme acti vity compared with the dimeric S-enzyme.