Amphiphilic Pig Intestinal Microvillus Maltase/Glucoamylase: Structure and Specificity

Abstract

Pig intestinal microvillus maltase/glucoamylase (EC 3.2.1.20) was solubilized from microvillus membranes using Triton X‐100 and was purified by a specific anti‐(maltase/glucoamylase) immunoadsorbent using elution with hypotonic solution. The specific activity was 20–35 U × mg−1 and the yield 11%. The enzyme preparations were free of other known microvillus disaccharidases and peptidases (less than 0.4%) and showed one precipitate in crossed immunoelectrophoresis. The Triton‐solubilized maltase/glucoamylase was amphiphilic as indicated by its detergent‐binding properties. These properties were abolished by treatment with papain and was not registered for the enzyme released from the microvillus membranes by papain. The Mr of the enzyme purified after solubilization by Triton X‐100 and papain was 330000 and 210000, respectively, as determined by gel filtration. The Mr, difference indicates that small‐molecular‐weight peptide(s) binding a Triton X‐100 micelle is (are) removed by proteolytic treatment. Maltase/glucoamylase isolated from pigs in which the pancreas had been completely disconnected from the duodenum three days before killing, migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as one polypeptide chain (Mr 245000–240000). This was also the only band seen in some preparations obtained from ordinary pigs. However, in preparations from normal pigs, most frequently two additional bands (Mr, 135000 and Mr 125000) with similar intensity to the larger band were seen. These two bands could be generated by treating preparations containing solely the larger band by pancreatic proteinases. Maltase/glucoamylase hydrolysis of maltose and starch had a broad pH optimum (pH 6–7). Using maltose as substrate the kinetics seemed to obey Michaelis‐Menten kinetics (Km 1.74 mM). The enzyme effectively cleaves off glucose residues from the nonreducing end of glucose polymers containing α‐(1→4) glucosidic bonds; α‐(1→6) bonds are also hydrolyzed but at a slow rate. The highest activities were observed for maltose and maltohexaose while the intermediate linear α(1→4) glucose polymers were hydrolyzed less effectively. Copyright © 1982, Wiley Blackwell. All rights reserved