Amphiphilic Pig Intestinal Microvillus Maltase/Glucoamylase

Abstract

1. Pig intestinal microvillus maltase/glucoamylase (EC 3.2.1.20) was solubilized from microvillus membranes using Triton X-I 00 and was purified by a specific anti-(maltase/glucoamylase) immunoadsorbent using elution with hypotonic solution. The specific activity was 20 -35 U x mg-' and the yield 13 %. The enzyme preparations were free of other known microvillus disaccharidases and peptidases (less than 0.4%) and showed one precipitate in crossed immunoelectrophoresis.2. 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 M , of the enzyme purified after solubilization by Triton X-100 and papain was 330000 and 210000, respectively, as determined by gel filtration. The M , difference indicates that small-molecular-weight peptide(s) binding a Triton X-100 micelle is (are) removed by proteolytic treatment.3. 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 ( M , . 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 ( M , 135 000 and M , 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.4. 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 (K, 3.74 mM). The enzyme effectively cleaves off glucose residues from the nonreducing end of glucose polymers containing a-(1 + 4) glucosidic bonds; cc-(1+6) bonds are also hydrolyzed but at a slow rate. The highest activities were observed for maltose and maltohexaose while the intermediate linear cc-(l+ 4) glucose polymers were hydrolyzed less effectively.Small intestinal maltase/glucoamylase (EC 3.2.1.20) is located in the microvillus membrane of the enterocytes [I, 21. The enzyme has earlier been purified as a papain-solubilized form from rat [3-61, rabbit [7] and human [8]. Papain solubilization probably releases the major, hydrophilic part of the enzyme carrying the activity, leaving the anchoring part in the membrane. Specificity studies on these enzyme preparations have revealed an a-glucosidase activity against glucose polymers of different size. The results on the structure of the papain-solubilized forms are difficult to interpret, as nicks may be introduced in the peptide chains during solubilization by the proteolysis.It has been demonstrated that maltase/glucoamylase can also be solubilized with the nonionic detergent Triton X-1 00 [9,10]. Detergent-solubilized maltase/glucoamylase was purified from rat intestine and some physica...

Biosynthesis of intestinal microvillar proteins

“…Whereas the activities of membrane-bound enzymes [aminopeptidase N (EC 3.4.11.2) and dipeptidy1 peptidase IV (EC 3.4.14.5)] could be partially recovered in the membrane fraction after the alkaline treatment (about 40%), they were totally absent in the supernatant. The exclusive presence in the membrane fraction of major bands of Mr 240000 and a blur of bands of M, 120000-150000, representing maltase-glucoamylase (EC 3.2.1.20), sucrase-isomaltase (EC 3.2.1.48-10) and aminopeptidase N [17,19,20] confirmed this finding ( fig. IA).…”

“…The membrane-bound polypeptides exhibited a different pattern. Pancreatic proteinases are known to cleave the most abundant microvillar enzymes in vivo [ 17,19,20]. Since labelling in organ culture was performed in the absence of pancreatic proteinases, the two lanes of membrane-bound polypeptides in fig.1 cannot be directly compared.…”

Biosynthesis of microvillar proteins

“…For pig sucraseisomaltase and maltase-glucoamylase, it has been shown that the extracellular proteolytic cleavage of the precursors has little, if any, effect on the specific activity (Sjostr6m et al, 1980;S0rensen et al, 1982), but it is unknown whether the same holds for the intracellular cleavage of y-glutamyl transpeptidase and lactase-phlorizin hydrolase. The importance of high-mannose glycosylation for biological activity has generally been found to vary considerably for various proteins (Gibson et al, 1980), but the drastic effect of tunicamycin (Danielsen & Cowell, 1984) suggests that it is essential for the correct folding of at least some microvillar enzymes.…”

On the primary site of control in the spontaneous development of small‐intestinal sucrase‐isomaltase after birth

“…before the appearance of SI mRNA, is, as mentioned in section 3, unrelated to the sucraseisomaltase complex and does not indicate an early, isolated synthesis of the isomaltase subunit alone (the cDNA probe used encompassed the whole of the isomaltase portion also). This early isomaltase activity is either due to a kindred enzyme, the glucoamylase complex (which is known to appear slightly ahead of sucrase-isomaltase [33f and may have some isomaltase activity [21][22][23][24][25]), or, more probably, to a lysosomal enzyme [32].…”