Purification and partial characterization of the high and low molecular weight form (S- and F-form) of invertase secreted by Aspergillus nidulans.
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 (pI 4.9-5.2) and the F-form (pI 3-4.2) were observed after isoelectric focusing. Deglycosylation with Endo H simplified this pattern to one enzymically active protein 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 than that of F-invertase both before and after deglycosylation. The Km 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.
Chen
J S
JS
Department of Biochemistry, University of Nottingham, UK.
Saxton
J
J
Hemming
F W
FW
Peberdy
J F
JF
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D006031:Glycosylation; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D043324:beta-Fructofuranosidase
D002241:Carbohydrates; D005656:Fungal Proteins; D007527:Isoenzymes; D010940:Plant Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase