Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papa?n solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.     

Phylogenetic analysis reveals key residues in substrate hydrolysis in the isomaltase domain of sucrase-isomaltase and its role in starch digestion

BackgroundStarch constitutes one of the main sources of nutrition in the human diet and is broken down through a number of stages of digestion. Small intestinal breakdown of starch-derived substrates occurs through the mechanisms of small intestinal brush border enzymes, maltase-glucoamylase and sucrase-isomaltase. These enzymes each contain two functional enzymatic domains, and though they share sequence and structural similarities due to their evolutionary conservation, they demonstrate distinct substrate preferences and catalytic efficiency. The N-terminal isomaltase domain of sucrase-isomaltase has a unique ability to actively hydrolyze isomaltose substrates in contrast to the sucrase, maltase and glucoamylase enzymes.MethodsThrough phylogenetic analysis, structural comparisons and mutagenesis, we were able to identify specific residues that play a role in the distinct substrate preference. Mutational analysis and comparison with wild-type activity provide evidence that this role is mediated in part by affecting interactions between the sucrase and isomaltase domains in the intact molecule.ResultsThe sequence analysis revealed three residues proposed to play key roles in isomaltase specificity. Mutational analysis provided evidence that these residues in isomaltase can also affect activity in the partner sucrase domain, suggesting a close interaction between the domains.Major conclusionsThe sucrase and isomaltase domains are closely interacting in the mature protein. The activity of each is affected by the presence of the other.General Significance: There has been little experimental evidence previously of the effects on activity of interactions between the sucrase-isomaltase enzyme domains. By extension, similar interactions might be expected in the other intestinal α-glucosidase, maltase-glucoamylase.     

Activity of renal hydrolases in pre- and postnatal development of mice

The fetal and postnatal activity patterns of different hydrolytic enzymes (alkaline phosphatase, gamma-glutamyltransferase, trehalase, maltase, glucoamylase, lactase, and sucrase) have been examined in mouse renal homogenates. Alkaline phosphatase and gamma-glutamyltransferase activities presented approximately similar changes. They increased from 18 days of gestation up to 30 days after birth. These activities showed marked increases during the 3rd and 4th postnatal weeks. A similar important rise was observed for trehalase activity at the end of the suckling period. Maltase activity increased gradually after birth. Traces of lactase, sucrase, and glucoamylase activities were detected at each developmental stage.     

Effects of hydrocortisone on carbohydrase concentrations, de novo synthesis and turnover patterns in immature rat intestine

Hydrocortisone administration to infant rats enhanced cellobiase and maltase activities and induced precocious expression of sucrase and trehalase activities along the length of the small intestine. These activity changes reflected proportional concentration increases in the enzymes lactase (EC 3.2.1.23), maltase/glucoamylase (EC 3.2.1.20) and sucrase-isomaltase (EC 3.2.1.48/10). Administration of an equivalent tracer dose of [3H]leucine (by body weight) to control and hydrocortisone-treated infant rats resulted in greater accumulation of label in the carbohydrase pools of the treated rats, suggesting their increased de novo synthesis. The increased concentrations of lactase and maltase/glucoamylase induced by exogenous hydrocortisone were matched by the presence of corresponding greater amounts of label in their brush border pools. Accumulation of label in each of the lactase, maltase/glucoamylase and sucrase-isomaltase pools was generally similar in the hydrocortisone-treated rats, suggesting equivalent stimulation of their synthesis as a group by the humoral agent. The turnover rates of the carbohydrases as a group were found to be similar and did not appear to differ in control and hydrocortisone-treated rats. Total protein synthesis rates were slightly greater in the intestine of the hydrocortisone-treated group of rats.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation.

The postition of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and gamma-glutamyl-transferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Intestinal lactase and other disaccharidase activities of a suckling crabeater seal (Lobodon carcinophagus)

1. The intestinal disaccharidase activities of a suckling crabeater seal were investigated. 2. Lactase, maltase, isomaltase and cellobiase activities were readily detected but trehalase and sucrase activities were absent. 3. The intestinal homogenates were separated into a soluble (S2) fraction and a particulate brush border (P2) fraction. The lactase activities of the two fractions had different properties corresponding to those of an acid and a neutral beta-galactosidase respectively. Approximately two-thirds of the total lactase activity measured at pH 6.0 was due to the acid beta-galactosidase. 4. The isomaltase and cellobiase activities were found almost exclusively in the particulate fractions but about one third of the maltase activity was in the S2 fraction. This soluble maltase activity appeared to be due to an acid maltase.     

Intestinal disaccharidases in the house musk shrew, Suncus murinus: occurrence of sucrase deficiency.

1. Disaccharidase activities of the small-intestinal brush border membrane were studied in six laboratory lines of the house musk shrew, Suncus murinus. 2. Sucrase activity was detected in all shrews of one line, but not in any shrew of three lines. In the other two lines it was found in some shrews, but not in the others. 3. Maltase, isomaltase, trehalase and lactase activities were found in all shrews of all the lines examined. 4. Sucrase was normally associated with isomaltase to form an enzyme complex. 5. Detergent-solubilized isomaltase, whether associated with sucrase or not, was inhibited by antibodies against rabbit sucrase-isomaltase to almost the same extent as the rabbit one, suggesting that isomaltase is not affected by a mutation(s) in sucrase.     

Intestinal brush border membrane-bound disaccharidases of the American alligator, Alligator mississippiensis

1. The disaccharidase activities of the small intestines of American alligators (Alligator mississippiensis) were studied in epithelial scrapes and brush-border membrane preparations. 2. Maltase, isomaltase and trehalase activities were found. Activities of these enzymes were higher in the proximal small intestine and decreased distally. 3. Disaccharidase activities were enriched 12-15 times in brush-border membrane preparations, compared with mucosa/enterocyte crude homogenates and were co-enriched with the brush-border membrane marker alkaline phosphatase. 3. The pH optima were: maltase 6.5; isomaltase 5.6; and trehalase 5.8. The Q10 of maltase, the most active enzyme, was equal to 1.82. 4. In reptiles, as in mammals, disaccharidase activities may be correlated with feeding habits. The co-occurrence of sucrase and isomaltase may not be a common feature of vertebrates.     

Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick

1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different K(m) values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose, palatinose and maltose. The second peak contained three disaccharidase enzymes all specific for the hydrolysis of maltose. 8. It is proposed that the intestinal disaccharidases of the chick exist in the form of two complexes: a sucrase-isomaltase complex and a maltase complex.     

The fetal and postnatal development of small intestinal disaccharidases in the rabbit

The development of small intestinal enzymes (lactase, acid- and hetero beta-galactosidases, cellobiase, maltase, trehalase, and sucrase) was studied from 18 days after conception until birth in 24 rabbit fetuses, and during the postnatal period in 15 newborn, juvenile, and adult rabbits. Lactase, acid- and hetero beta-galactosidases, cellobiase, and trehalase activities increased significantly during the fetal stage, while changes in sucrase and maltase activities were not substantial. In the postnatal period, lactase and cellobiase activities decreased significantly whereas maltase, sucrase, and trehalase activities increased significantly to reach adult values by 30 days of age. The acid- and hetero beta-galactosidases remained unchanged.     

Inhibition of beta-fructofuranosidases and alpha-glucosidases by synthetic thio-fructofuranoside

A synthetic beta-thio-fructofuranoside of mercaptoethanol inhibited not only beta-fructofuranosidases but also alpha-glucosidases. The compound was hardly hydrolyzed by the glycosidases. The thio-fructoside competitively inhibited beta-fructofuranosidases from Aspergillus niger, Candida sp., and Saccharomyces cerevisiae, but not Arthrobacter beta-fructofuranosidase at all. Sucrase activity of rat intestinal sucrase/isomaltase complex was also suppressed in the presence of the thio-fructoside. The thio-fructoside showed noncompetitive inhibition toward maltase activity of the rat intestinal enzyme complex and Saccharomyces sp. alpha-glucosidase. Inhibition against the Bacillus stearothermophilus alpha-glucosidase, Rhizopus glucoamylase, and porcine kidney trehalase were more slight than that against these two alpha-glucosidases.     

Insulin accelerates the development of intestinal brush border hydrolytic activities of suckling mice

The influence of insulin on the postnatal development of intestinal brush border membrane disaccharidase (sucrase, maltase, trehalase, lactase) and peptidase (leucylnaphthylamidase, γ-glutamyltranspeptidase) activities has been studied in mice. At 8 days of age, the animals received either 5, 10, or 12.5 mU insulin/g body wt/day during 3 days. A premature appearance of sucrase activity was noted, the level of sucrase activity being dependent of the amount of insulin injected. Maltase and lactase activities were both increased while trehalase activity was affected only by the highest dose of insulin. The behavior of the two peptidases was quite different as γ-glutamyltranspeptidase was prematurely increased and leucylnaphthylamidase was unaffected by insulin. The hormonal effect is exerted along the entire small intestine. The time course of the responses of the disaccharidases in relationship to cellular migration along the crypt-villus axis has also been studied. By 24 hr after administration of a single injection of 12.5 mU insulin/g body wt, sucrase activity was already present and an increased maltase and trehalase activities were observed. During the subsequent 72 hr no further increase of enzymatic activity was noted even though the epithelial cells are moving up on the villi at a faster rate than in controls, thus indicating that there is no relationship between the enzymatic responses and the cellular migration. The present data show that a premature increase of the circulating level of insulin influences the development of intestinal mucosa in suckling mouse.     

Intestinal lactase (beta-galactosidase) and other glycosidase activities in suckling and adult tammar wallabies (Macropus eugenii)

The activities of various glycosidases in homogenates of the small intestinal mucosa of two adult and 18 suckling tammar wallabies (M. eugenii) aged from 6 to 50 weeks were investigated. Lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, alpha-L-fucosidase and neuraminidase activities were high during the first 34 weeks post partum and then declined to very low levels. Maltase, isomaltase, sucrase and trehalase activities were very low or absent during the first 34 weeks, and then increased. The lactase activity was unusual in being greater in the distal than the middle or proximal thirds of the intestine, and in its low pH optimum (pH 4.6), inhibition by p-chloromercuribenzene sulfonate but not by Tris, and lack of cellobiase activity. These properties are those of a lysosomal acid beta-galactosidase rather than of a brush border neutral lactase. The maltase activity had the characteristics of a lysosomal acid alpha-glucosidase early in lactation and of a brush border neutral maltase in adult animals. The significance of these findings is discussed in relation to changes in dietary carbohydrates during weaning and to the mode of digestion of milk carbohydrates by the pouch young.     

The level and distribution of disaccharidases in the camel (Camelus dromedarius) intestine

1. The disaccharidases, cellobiase, isomaltase, lactase, maltase, sucrase and trehalase were investigated for presence in the camel (Camelus dromedarius) intestine and pancreas. All, except sucrase, were present. 2. Their levels of activities were measured at different positions of the small and large intestines and the location of maximum level of activity for each enzymes along the intestinal tract was established. 3. High levels of activities were determined in the contents of the intestinal lumen and, therefore, it is absorbed into the cells of the epithelial villi and hydrolyzed there. 4. The possibility of carbohydrate digestion in camel intestine is discussed.     

Characterization of nimbidiol as a potent intestinal disaccharidase and glucoamylase inhibitor present in Azadirachta indica (neem) useful for the treatment of diabetes

Azadirachta indica, used in antidiabetic herbal drugs, was reported to contain α-glucosidase inhibitor. Bioassay guided purification characterized the inhibitor as nimbidiol (a diterpenoid), present in root and stem-bark of the tree. Nimbidiol inhibited intestinal (mammalian) maltase-glucoamylase, sucrase-isomaltase, lactase, trehalase and fungal α-glucosidases. Nimbidiol showed a mixed competitive inhibition on intestinal carbohydrases. IC₅₀, Ki and Ki′ (µM) were 1.35 ± 0.12, 0.08 ± 0.01, 0.25 ± 0.11, respectively, for maltase-glucoamylase (maltotetraose as substrate). Nimbidiol was more potent inhibitor of isomaltase (IC₅₀ 0.85 ± 0.035 µM), lactase (IC₅₀ 20 ± 1.33 µM) and trehalase (IC₅₀ 30 ± 1.75 µM) than acarbose, voglibose, salacinol, kotalanol and mangiferin. Ki and Ki′ values (µM) for intestinal sucrase were 0.7 ± 0.12 and 1.44 ± 0.65, respectively. Development of nimbidiol as an antidiabetic drug appears to be promising because of broad inhibition spectrum of intestinal glucosidases and easy synthesis of the molecule.     

Inhibitory effects of ellagi- and gallotannins on rat intestinal alpha-glucosidase complexes

The clove ellagitannins and their related polygalloyl-glucoses inhibited maltase activity of rat intestinal alpha-glucosidases. The structure-activity relationship study of those galloylglucoses, varying the extent of galloylation on the glucose core, with the ellagitannins, indicated that an increasing number of galloyl units in the molecule lead to an increase in the inhibitory activity. Penta-O-galloyl-beta-D-glucose, with five galloyl groups showed the highest inhibitory activity. On the other hand, hexahydroxydiphenoyl units contained in the ellagitannins had little effect on the activity. After separation of maltase-glucoamylase and sucrase-isomaltase complexes from the crude mixture of the rat alpha-glucosidases, the inhibitory activities of the galloylglucose derivatives against each complex were examined. The inhibitory influence on the maltase-glucoamylase complex was more potent than on the sucrase-isomaltase complex.     

Evidence for a possible regulatory gene (Suc-1) controlling sucrase expression in mouse intestine

Assays for sucrase carried out on intestinal sonicates prepared from 18 different strains of mice revealed a threefold variation in specific activity, the values for CBA/Ca mice being significantly less than for any other strain. Further comparison of the CBA/Ca versus the C57BL/6J mouse showed this deficiency, which became established 2–4 weeks after birth, to apply to isomaltase as well as sucrase but not to maltase or trehalase. Backcross experiments indicated that this deficiency in sucrase activity was inherited as a single codominantly expressed genetic factor. The ability of the CBA/Ca mouse to regulate sucrase activity in response to changes in diet was also reduced compared to that of the C57BL/6J mouse. No difference could be detected in the affinity of sucrase for its substrate or in the ability of heat to denature sucrase prepared from CBA/Ca and C57BL/6J mice. It is suggested that part of the regulatory region of the gene coding for sucrase-isomaltase is modified in the CBA/Ca mouse and that this locus should be given the notation Suc-1 for future reference.This work supported by an MRC project grant to M. W. Smith.