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Studies on the size and shape of rabbit intestinal glucoamylase-maltase complex.

ABSTRACT: Rabbit intestinal glucoamylase-maltase was examined in detail with respect to its molecular weight, sedimentation, diffusion and viscosity. It is a large asymmetrical molecule, with a molecular weight of 750 000-760 000. Its appearance under the electron microscope supports the idea that it is a long string (62.0 nm) consisting of eight beads of diameter 6.0 nm each and a surface-to-surface interbead distance of approx. 2.0 nm. The shape of the enzyme derived from its hydrodynamic behaviour by using the string-of-spherical-beads model originally proposed by Kuhn [(1932) Z. Phys. Chem. Abt. A 161, 1-32] and later modified by Shulman [(1953) J. Am. Chem. Soc. 75, 5846-5852] fits moderately well with the electron-microscopic picture. The beads might represent about six subunits, and the absence of sulphur from the enzyme and the inability to dissociate the enzyme by conventional methods indicate the possibility of unusual covalent cross-linking between the subunits and between the beads.

SUBMITTER: Sankaran K

PROVIDER: S-EPMC1152188 | biostudies-other | 1983 Sep

REPOSITORIES: biostudies-other

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Structural insight into substrate specificity of human intestinal maltase-glucoamylase.

Project description:Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.

| S-EPMC4875297 | biostudies-literature

Neutral maltase/glucoamylase from rabbit renal cortex.

Project description:Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.

| S-EPMC1138778 | biostudies-other

The effect of polyhydroxylated alkaloids on maltase-glucoamylase.

Project description:One of the most important carbohydrate-splitting enzymes is themaltase-glucoamylase which catalyzes the hydrolysis of alpha-glucosidic linkages. Maltase-glucoamylase inhibitors during the last few years have aroused medical interests in the treatment of diabetes. They contribute to a better understanding of the mechanism of maltase-glucoamylase. At present there are many different classes of maltase-glucoamylase inhibitors. This paper focuses on alkaloidal inhibitors of maltase-glucoamylase and structure-activity relationship (SAR) studies between them in order to discover some drugs with better efficiency and lower toxicity for treating diabetes.

| S-EPMC3742645 | biostudies-literature

Enzyme activity in partly dissociated fragments of rat intestinal maltase/glucoamylase.

Project description:Pure rat intestinal maltase/glucoamylase was partially inactivated in 1% sodium dodecyl sulphage by heating at 40--70 degree C for 5 min at pH 7.5, or by lowering the pH to 5.4--6.6 at 24 degree C. When partially active preparations were electrophoresed in the presence of sodium dodecyl sulphate, a complicated protein band pattern of incompletely dissociated fragments of the enzyme was observed. Complete dissociation of the enzyme in sodium dodecyl sulphate, induced by boiling or by pH values below 5.4, was accompanied by total loss of enzyme activity and simplification of the protein pattern to five major species. Although the original enzyme band was absent from some partially dissociated preparations, enzyme activity was present and was associated with several transient protein bands on the gels. Maltase and alpha-glucosidase activities were detected in these bands, but glucoamylase activity was absent.

| S-EPMC1186398 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV.

Project description:The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

| S-EPMC1154236 | biostudies-other

The maltase, glucoamylase and transglucosylase activities of acid -glucosidase from rabbit muscle.

Project description:1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.

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The maltase-glucoamylase gene: common ancestry to sucrase-isomaltase with complementary starch digestion activities.

Project description:Brush-border maltase-glucoamylase (MGA) activity serves as the final step of small intestinal digestion of linear regions of dietary starch to glucose. Brush-border sucrase-isomaltase (SI) activity is complementary, through digestion of branched starch linkages. Here we report the cloning and sequencing of human MGA gene and demonstrate its close evolutionary relationship to SI. The gene is approximately 82,000 bp long and located at chromosome 7q34. Forty-eight exons were identified. The 5' gene product, when expressed as the N-terminal protein sequence, hydrolyzes maltose and starch, but not sucrose, and is thus distinct from SI. The catalytic residue was identified by mutation of an aspartic acid and was found to be identical with that described for SI. The exon structures of MGA and SI were identical. This homology of genomic structure is even more impressive than the previously reported 59% amino acid sequence identity. The shared exon structures and peptide domains, including proton donors, suggest that MGA and SI evolved by duplication of an ancestral gene, which itself had already undergone tandem gene duplication. The complementary human enzyme activities allow digestion of the starches of plant origin that make up two-thirds of most diets.

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Interaction between the α-glucosidases, sucrase-isomaltase and maltase-glucoamylase, in human intestinal brush border membranes and its potential impact on disaccharide digestion.

Project description:The two major intestinal α-glycosidases, sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM), are active towards α-1,4 glycosidic linkages that prevail in starch. These enzymes share striking structural similarities and follow similar biosynthetic pathways. It has been hypothesized that starch digestion can be modulated via "toggling" of activities of these mucosal α-glycosidases, suggesting a possible interaction between these two enzyme complexes in the intestinal brush border membrane (BBM). Here, the potential interaction between SI and MGAM was investigated in solubilized BBMs utilizing reciprocal pull down assays, i.e., immunoprecipitation with anti-SI antibody followed by Western blotting with anti-MGAM antibody and vice versa. Our results demonstrate that SI interacts avidly with MGAM concomitant with a hetero-complex assembly in the BBMs. This interaction is resistant to detergents, such as Triton X-100 or Triton X-100 in combination with sodium deoxycholate. By contrast, inclusion of sodium deoxycholate into the solubilization buffer reduces the enzymatic activities towards sucrose and maltose substantially, most likely due to alterations in the quaternary structure of either enzyme. In view of their interaction, SI and MGAM regulate the final steps in starch digestion in the intestine, whereby SI assumes the major role by virtue of its predominant expression in the intestinal BBMs, while MGAM acts in auxiliary supportive fashion. These findings will help understand the pathophysiology of carbohydrate malabsorption in functional gastrointestinal disorders, particularly in irritable bowel syndrome, in which gene variants of SI are implicated.

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Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

Project description:Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.

| S-EPMC2878540 | biostudies-literature

Kinetic studies on glucoamylase of rabbit small intestine.

Project description:The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with alpha-1,4 and alpha-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.

| S-EPMC1172577 | biostudies-other

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