Some properties of monkey intestinal sucrase

A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen.     

The mechanism of the human intestinal sucrase action

Highly purified sucrase accepts as a substrate α-anomers within D-glucopyranosides. Hydrolysis of sucrose and palatinose processds with the net retention of configuration at the C-1 of D-glucopyranose. The fission of the glycosidic bond takes place between the C-1 of the D-glucose ring and the glycosidic oxygen; therefore D-fructose is released as β-D-fructofuranose from sucrose. During hydrolysis, transglycosylation takes place and a few oligosaccharides are formed and in the presence of methanol α-methyl-D-glucopyranoside is also produced. Non-stoichiometric amounts of D-glucose and D-fructose are produced from sucrose, turanose and palatinose and less D-glucose than expected is present. Formation of sucrose from D-glucose and D-fructose was not observed.     

Blood group activity of human sucrase from intestinal metaplasia.

Sucrases were purified from human small intestine and from areas of intestinal metaplasia of the stomach mucosa surrounding stomach cancers. The kinetic constants and pH activity profiles of enzyme preparations from the two sources were similar. No blood group activity of sucrase was detectable in preparations from three cases of intestinal metaplasia, but preparations from two other cases showed activity like that of the small intestine. These results indicate that sucrase from areas of intestinal metaplasia has similar enzymatic properties to those of enzyme from the small intestine, but that the antigenic sugar moiety of the enzyme associated with blood group activity varies.     

Purification and partial characterization of chick intestinal sucrase

The sucrase was purified from the small intestinal mucosa of the adult chick. Purification procedure involved solubilization with papain, ethanol precipitation, chromatography on Sephadex G-200 and DEAE-Sephadex. Several characters of the chick intestinal sucrase resembled those of the intestinal sucrase-isomaltase complex of some mammals (rabbit, rat and human).     

Insulin-evoked precocious appearance of intestinal sucrase activity in suckling mice.

The effect of insulin (12.5 mU/g body wt/day) on the ontogeny of intestinal sucrase has been studied in suckling mice. Sucrase activity normally appears along the entire small intestine between the 14th and 16th days after birth. The hormonal treatments begin at 8 days and the response of sucrase to one or three injections of hormone is subsequently analyzed in the proximal, middle, and distal intestinal thirds. Three injections of insulin provoke a precocious appearance of sucrase in all intestinal parts, the proximal third exhibiting the highest sucrase activity. Twenty-four hours after a single injection of insulin, sucrase activity can already be detected along the entire small intestine. During the second and third days, the activities observed in the different parts of the small intestine remain stable. These data show that insulin is able to provoke a premature appearance of sucrase activity and appears to play a previously unsuspected role in intestinal maturation.     

Inhibition of porcine small intestinal sucrase by valienamine

Valienamine, an aminocyclitol, has been isolated from the enzymolysis broth of validamycins. The absolute configuration of valienamine is similar to that of alpha-D-glucose. The inhibitory effect of this amino-sugar analog of alpha-D-glucose, valienamine, on porcine small intestinal sucrase was examined. Valienamine was found to be potent, competitive reversible inhibitor of porcine small intestinal sucrase in vitro with an IC50 value of 1.17 x 10(-3)M. Valienamine also exhibited dose-dependent, instantaneous inhibition of porcine small intestinal sucrase. The inhibition of porcine small intestinal sucrase by valienamine was pH-independent.     

Harmaline interaction with sodium-binding sites in intestinal brush border sucrase.

The effect of harmaline on rabbit brush border sucrase has been studied at pH 6.8. An initial analysis in classical kinetic terms revealed harmaline to be a fully competitive inhibitor of the substrate, sucrose. In spite of this result however, the following hypothesis has been tested. Harmaline, which is positively charged in the physiological range of pH, might in fact compete, not directly with the substrate site, but rather with an allosterically-related sodium-binding site which has been postulated to be involved in the activation of sucrase by the alkali-metal ions (Mahmood and Alvarado, Arch. Biochem. Biophys. 168, 585, 1975). Because of its size, harmaline, when bound to the metal site, could at least partially overlap with the substrate site, thereby behaving as if it were an authentic fully competitive inhibitor of the substrate. This hypothesis appears to be confirmed by the fact that the alkali metals can completely reverse the inhibition caused by harmaline.     

A probable oxocarbonium ion in the reaction mechanism of small intestinal sucrase and isomaltase.

1-5-D-Gluconolactone is a competitive inhibitor of both sucrase and isomaltase. Substitution of the 1H and 2H at C1 of the glucosyl moiety in p-CL-phenyl-alpha-D-glucopyranoside leads to a decrease in kcat of both sucrase and isomaltase, the k1H/k2H ranging between 1.14 and 1.20. Treatment of the association constants and of the kcat values for a number of p-substituted phenyl-alpha-D-glucopyranosides on the basis of the Hammet-Hansch equation has allowed the estimation of the importance of hydrophilicity-hydrophobicity as well as of the magnitude of the p values for both substrate-enzyme interaction and catalysis in both sucrase and isomaltase. The magnitude of the secondary deuterium effect as well as the low values of p in both sucrase and isomlatase are strongly indicative of the rate-limiting step going through the formation of an oxocarbonium ion. In conjunction with other observations reported previously, the data presented here led to the suggestion of the main lines of a reaction mechanism for the two glucosidases: prptonation of the glycosidic oxygen is followed by the liberation of the "aglycone" with formation of an oxocarbonium ion, which is temporarily stabilized by a carboxylate group.     

Small intestinal sucrase and isomaltase split the bond between glucosyl-C1 and the glycosyl oxygen.

The products of the hydrolysis of sucrose and palatinose by the sucrase-isomaltase complex from rabbit small intestine were investigated by persilylation followed by gas-liquid chromatography and mass spectrometry. If the hydrolysis is carried out in H218O, the heavy oxygen is found exclusively at the Ci of the alpha-glucopyranose formed. The 18O enrichment equals that of the incubation medium. The oxygen exchange between the monosaccharides and water is not accelerated by the sucrase-isomaltase complex. These observations show that the bond split by the sucarse and the isomaltase moiety of the complex is the one between glucosyl-Ci and the glucosyl oxygen. They agree with the mechanism proposed for these carbohydrases in the accompanying paper (Cogoli, A., and Semenza, G. (1975) J. Biol. Chem. 250, 7802-7809) involving the formation of an oxocarbonium ion.     

Rat intestinal sucrase inhibition of constituents from the roots of Rosa rugosa Thunb.

A new octanordammarane triterpene, 3β,15α-dihydroxymansumbinol (1) and a novel A-ring contracted oleanane triterpenoid, 2-formyl-(A)1–19α-hydroxy-1-norolean-2,12-dien-28-oic acid (2) were isolated from the roots extract of Rosa rugosa along with fifteen known compounds (3–17). Their structures were elucidated by extensive spectroscopic analysis, including 1D and 2D NMR, and FTICRMS. The MeOH extract, as well as CH₂Cl₂ and EtOAc fractions at a concentration of 0.5 mg/mL showed potent sucrase inhibitory activity, with inhibition percentage values of 84.67 ± 5.37%, 87.50 ± 2.78%, and 81.91 ± 2.90%, respectively. In addition, compounds 7–13 (1.0 mM) showed potent sucrase inhibitory activity (61.88 ± 3.19% to 84.70 ± 3.07% inhibition), which was comparable to that of the positive control, acarbose, with an inhibition percentage value of 50.96 ± 2.97%. Compounds 1, 2, 4, and 14–17 showed moderate and/or weak inhibitory activities at the same concentration. The α-glucosidase inhibitory activities of the extracts and purified compounds may provide a novel opportunity to develop a new class of antidiabetic agents.