Abstract
Goose (Anser anser) kidney microvillus sucrase-isomaltase (EC 3.2.1.48-EC3.21.10) was solubilized from isolated microvillus membranes using Emulphogen BC 720 or papain. Detergent-solubilized enzyme (D-SI) was purified 149±29 times with a yield of 15.7±2.6% by a two-step procedure which included chromatofocusing. The specific activity was 2.95±0.34 U/mg protein for sucrase, 1.02±0.13 for palatinase and 5.63±0.53 for maltase. D-SI was amphiphilic as indicated by its detergent-binding properties. These properties were not observed for sucrase-isomaltase released from the microvillus membrane by papain. The M r of the enzyme purified after solubilization by Emulphogen and papain was 543 000 and 380 000, respectively, as determined by gel filtration. The difference in M r indicates that an Emulphogen micelle is bound to the detergent-solubilized enzyme. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sucrase-isomaltase migrated as several polypeptide chains: a major band ( M r 280 000) and at least seven additional minor bands ( M r 220 000-100 000). It is suggested that the major band represents the precursor pro-sucrase-isomaltase and that the lower molecular weight bands are generated by PMSF or aprotinin-resistant proteinases during homogenisation and chromatography of the enzyme. Measured by chromatofocusing, the isoelectric point was found to be pH 4.6. Sucrase-isomaltase accounts for about 20% of total microvillus membrane proteins.
Published Version
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