Purification and partial characterization of the high and low molecular weight form (S- and F-form) of invertase secreted by Aspergillus nidulans

• Published in: Biochimica et biophysica acta 1996-09, Vol.1296 (2), p.207-218
• Main Authors: Chen, Jee-song, Saxton, Janice, Hemming, Frank W., Peberdy, John F.
• Format: Article
• Language: English
• Subjects: A. nidulans; Amino Acid Sequence; Aspergillus nidulans - enzymology; beta-Fructofuranosidase; Carbohydrates - analysis; Characterization; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Extracellular enzyme; Fungal Proteins - metabolism; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Glycosylation; Invertase (S- and F-form); Isoenzymes - chemistry; Isoenzymes - isolation & purification; Molecular Sequence Data; Plant Proteins - chemistry; Purification; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/0167-4838(96)00073-8

Two forms of secreted invertase have been purified from Aspergillus nidulans by ion-exchange and gel-filtration chromatography. S-invertase gave a single, broad, glycoprotein band on PAGE and SDS-PAGE corresponding 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 mainly galactose (29%) and less mannose (12%). Three sharp bands of enzymically active glycoprotein for both the S-form (p I 4.9–5.2) and the F-form (p I 3–4.2) were observed after isoelectric focusing. Deglycosylation with Endo H simplified this pattern to one enzymically active protein band (p I 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 than that of F-invertase both before and after deglycosylation. The K m values of the two forms of invertase were very similar. Significant homology existed between the N-terminal amino-acid sequences of S-invertase (and of internal peptides derived from it) and sequences of invertase from other species. It is suggested that the higher carbohydrate content in F-invertase results in the native enzyme existing as a monomer and having a greater negative charge and lower specific enzyme activity compared with the dimeric S-enzyme.