Hydrolysis Of Maltose Research Articles

Animal experiment studies on parenteral utilization of maltose

The utilisation of parenterally administered maltose was investigated in the anaesthetized rat, and in rats fixed in metabolic cages. Additionally, the metabolism of maltose was measured with the isolated perfused rat liver. During intravenous infusion of 0.3 g, 0.6 g or 1.2 g maltose (corresponding to 0.9 g, 1.8 g or 3.6 g/kg bodyweight) per hour a steady-state for maltose in blood was attained. Blood glucose concentration rose during the maltose infusions. The utilisation of parenterally administered maltose was established by a high rate of glycogen storage in the liver and by a decrease in concentration of free fatty acids in serum. During the 72 hour infusion of maltose at a rate of 0.23 g/hour (corresponding to 0.70 g/kg bodyweight) a constant blood maltose concentration of 60 mg/100 ml was measured. Simultaneously, the blood glucose concentration increased. The excretion of maltose and of glucose was approximately 5% of the total amount administered intravenously. The nitrogen sparing effect of maltose was equal to that of glucose (or glucose substitutes). In the streptozotocindiabetic rats, the renal excretion of maltose and glucose was 20-30% of the total amount. Moreover, blood glucose concentration was elevated significantly during maltose infusion. In the isolated perfused rat liver maltose hydrolysis was established. However, the glucose obtained by this hydrolysis was not metabolized by the isolated organ as was observed for glucose. On the other hand, the glucose substitutes (fructose, xylitol, sorbitol) are also partially transformed to glucose by the isolated liver. These substances, however, were additionally utilized by this organ in other ways. On the basis of these results it is concluded that maltose is metabolized following its intravenous application. Therefore, maltose should have advantages compared to glucose because of the lower osmotic pressure. The metabolism of maltose, however, is similar to that of glucose. The advantages of the glucose substitutes (fructose, sorbitol, xylitol) are not shared by maltose.

Improved method for purification of buckwheat .ALPHA.-glucosidase and some kinetic properties.

An α-glucosidase which was homogeneous in disc electrophoretic and ultracentrifugal analysis was obtained from buckwheat seeds by fractionation with ammonium sulfate, chromatography on CM-cellulose and gel filtration on Bio-Gel P-150. The sedimentation coefficient (s20, w) was estimated to be 6.2 S, and the molecular weight, 80,000~88,000. The ratio of initial velocity of hydrolysis for maltose (Km = 2.13 mg/ml) and soluble starch (Km = 7.46 mg/ml) was calculated to be 100:86. Both activities were competitively inhibited by Tris and erythritol, and stimulated approximately 1.5-fold in the presence of 25 mm-K+ or Na+. The Ki values for Tris on hydrolysis of maltose and soluble starch were 0.78 mm and 1.07 mm, and those for erythritol, 77.7 mm and 64 mm, respectively.

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.

Partial purification and characterization of alpha-glucosidase from Pseudomonas fluorescens W

The alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) of Pseudomonas fluorescens W was partially purified by (NH4)2SO4 fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. The enzyme showed great specificity for maltose hydrolysis, with very little action against polymeric forms. Sucrose, isomaltose, alpha-methylglucoside, and maltobionic acid were not hydrolyzed. Turanose was a strong competitive inhibitor, and glucose a weaker one. Tris (2-amino-2-hydroxymethylpropan-1:3-diol) inhibited enzyme activity significantly only at alkaline pH. Mercuric, cupric, and silver cations strongly inhibited, and EDTA (ethylenediaminetetraacetate) weakly inhibited the enzyme. The isolated enzyme was rather unstable even at 4 degrees C, and was destroyed by freezing and lyophilization. Inositol and albumin had a slightly protective effect. Sulfhydryl-binding reagents strongly inhibited the enzyme.

Substrate concentration dependence of the rate of maltose hydrolysis by saccharifying alpha-amylase from B. subtilis.

1. The action of saccharifying alpha-amylase from B. subtilis [EC 3.2.1.1] on eq- (the equilibrium mixture of alpha- and beta-forms) and beta-maltose was studied at pH 5.4 and 25 degrees. 2. The plots of initial velocity versus substrate concentration showed remarkable sigmoidal curves for both substrates. 3. The value of Hill's coefficient for eq- and beta-maltose were determined to be 3.3 and 3.8, respectively.

Disaccharide hydrolysis, intestinal absorption and electrogenic properties in salmonella enterocolitis in mice.

The hydrolysis of maltose, sucrose or lactose and absorption of the corresponding monosaccharides using everted in vitro small intestine of mice heavily infected with Salmonella enteritidis var. chaco was significantly reduced compared with uninfected controls. Maltose was most efficiently hydrolyzed, sucrose less so, and lactose barely hydrolyzed. The serosal fluid transfer and gut fluid uptake generally decreased in the infected intestine. The absorption of free monosaccharides was also markedly reduced in the diseased intestine, and the serosal fluid transfer and gut fluid uptake decreased. Glucose and fructose were extensively metabolized to lactate during transfer in both the normal and diseased intestine, fructose apparently remaining unabsorbed. Electrical potential differences were associated with the rate of monosaccharide(s) absorption, and decreased in the infected intestine.

Brush border disaccharidases in dog kidney and their spatial relationship to glucose transport receptors.

The localization of disaccharidases in kidney has been studied by means of the multiple indicator dilution technique. A pulse injection of a solution containing Evans blue dye (plasma marker), creatinine (extracellular marker), and a (14)C-labeled disaccharide (lactose, sucrose, maltose, and alphaalpha-trehalose), is made into the renal artery of an anesthetized dog, and the outflow curves are monitored simultaneously from renal venous and urine effluents. Lactose and sucrose have an extracellular distribution. Trehalose and maltose remain extracellular from the postglomerular circulation. About 75% of filtered tracer maltose or trehalose is extracted by the luminal surface of the nephron. Thin-layer chromatography of urine samples shows that all of the excreted (14)C radiolabel is in the form of the injected disaccharide. Following the administration of phlorizin, all of the filtered radioactivity is recoverable in the urine, but chromatography of the urine samples now reveals that there is a significant excretion of [(14)C]glucose, approximating the amount previously extracted under control conditions (in the absence of phlorizin). It has been verified that no hydrolysis of maltose or trehalose to their constituent glucose subunits occurred during the transit of tracer between the point of injection (renal artery), and the point of filtration (glomerular basement membrane). Similarly, after addition of [(14)C]disaccharides to fresh urine there is no chromatographically recoverable [(14)C]glucose. It is concluded that there exist alpha-glucosidases with maltase and trehalase activity along the brush border of the proximal tubule and that these disaccharidases are located spatially superficial to the glucose transport receptors.

Carbohydrates of the bovine small intestine.

1. Homogenates of mucosa from bovine small intestine hydrolysed isomaltose slowly.2. The enzyme exhibited maximum activity at pH 6·0–6·2 and possessed aKmvalue of 9·1 mM.3. Isomaltase and maltase activities showed a similar pattern of distribution along the small intestine; activity was higher in the jejunum than in the ileum, the lowest activity being in the duodenum.4. Neither activity changed markedly with age.5. The disaccharidase activities (maltase, isomaltase, trehalase, lactase and cellobiase) of the small intestine were found in the sediment when homogenates were centrifuged at 100000g.6. Heat inactivation studies suggested that there was one enzyme which hydrolysed trehalose, one enzyme which hydrolysed isomaltose, one enzyme which hydrolysed lactose and cellobiose and that the hydrolysis of maltose was brought about by more than one enzyme.

A Simple Method to Differentiate between α- and β-Amylase

Physical and chemical properties often have been used to differentiate a-amylase from /3-amylase (2-4, 7). a-Amylase is insensitive to high temperatures and heavy metal ions, requires Ca2+ for activity, and is inactivated at a low pH. /8-Amylase is sensitive to high temperatures and heavy metal ions, does not require Ca2+, and is stable at a low pH. Enzymatic differences between the two forms of amylase have been used to a lesser extent. One enzymatic characteristic that has been used is the dissimilarity of the end products of amylolytic hydrolysis (3, 7). /3-Amylase hydrolyzes soluble starch or amylose, yielding only maltose as an end product. Hydrolysis of soluble starch or amylose by a-amylase occurs randomly, releasing a large amount of oligosaccharides or dextrin. The dextrin is then hydrolyzed, yielding a large quantity of maltose and little glucose and maltotriose (1). At this point, hydrolysis by aor 3-amylase would yield essentially similar end products. If the further hydrolysis of maltose by a-amylase is rate-limiting and is dependent upon the concentration of the enzyme and pH (7), little or no glucose would be expected. As a result, the chromatographic display of the end products would be similar to that obtained from /3-amylase hydrolysis.

Multiple Forms of α-Glucosidase From Bovine Spleen

α-Glucosidase (α-d-glucoside glucohydrolasc; EC: 3.2.1.20) has been extracted from bovine spleen and separated into two fractions (Fractions A and B) by gel filtration on Sephadex G-200. Fraction B which was retarded on Sephadcx columns contained only an acid α-glucosidase. This enzyme catalyzed hydrolysis of maltose and glycogen at comparable rates. Glycogen was hydrolyzed almost completely to glucose. The results of heat-treatment and of inhibition bv turanose demonstrated that Fraction A contained both acid if and neutral α-glucosidases. The neutral enzyme in Fraction A was further separated into four different components on preparative polyacrylamide gel electrophoresis. All of these neutral enzymes showed similar catalytic properties each other, and hydrolyzed maltose much more rapidly than glycogen. The acid enzyme in Fraction A was inactivated on the electrophoresis and was not further characterized.

Acid maltase levels in muscle in heterozygous acid maltase deficiency and in non-weak and neuromuscular disease controls.

Acid maltase (AM) deficiency carriers can be detected by muscle enzyme assay. The assay indicates that, just as in infantile and childhood cases, adult cases of the disease are transmitted by autosomal recessive inheritance. With the maltose hydrolysis assay, in some neuromuscular diseases, muscle AM activity can be as low as in heterozygous AM deficiency. A relatively low muscle AM activity in myxoedema myopathy is confirmed. In human muscle, the K(m) of the enzyme for maltose hydrolysis is 7·2 to 9 × 10(-3)M. A modification of the enzyme assay based on this fact is recommended.

Disaccharidase activity in the intestine of Ascaris lumbricoides Linné, 1758

1. 1. The luminal side of the Ascaris intestinal cells has maltase, but no lactase activity. 2. 2. When intestinal sacs containing maltose are incubated, high concentrations of glucose appear in the lumen. The mechanism of glucose transport saturates before the process of maltose hydrolysis. 3. 3. When intestinal sacs are incubated with sucrose, fructose appears in the lumen at higher concentrations than glucose. With sucrose and phloridzin, the concentration of luminal glucose equals the concentration of fructose. 4. 4. These experiments suggest that the hydrolysis of disaccharides and the transport of monosaccharides are independent processes and that only the latter can enter the cell.

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.

Kinetics and Mechanism of Transfer Action of Saccharifying α-Amylase of Bacillus subtilis

1. The formation of phenol (φ) was observed in the mixture of maltose (M) and phenyl α-glucoside (φG), which was catalyzed by saccharifying α-amylase (EC 3. 2. 1. 1) of Bacillus subtilis, although φG itself was not hydrolyzed either in the presence or absence of glucose. Thin layer chromatography (TLC) of the reaction mixture revealed the formation of a small amount of phenyl α-maltoside (φM). From these results, it was concluded that φM was produced from M and φG by glucosyl transfer catalyzed by the enzyme, and then φM was hydrolyzed into φ and M. Since the overall rate of formation of phenol, vφ, is limited by the rate of transfer, vr, the latter can be obtained directory from the measurement of the former. This system was utilized for studying the kinetics of transfer action of this enzyme. 2. The rate of transfer as a function of φG concentration at a fixed concentration of M proceeded according to the Michaelis-Menten kinetics within the range of concentrations of φG examined, and the apparent Michaelis constant KmφG for φG was 250 mM at a maltose concentration of 42 mM. On the other hand, the rate of transfer, vr, as a function of the concentration of M in a fixed concentration of φG showed a remarkable sigmoidal shape in the region of lower substrate concentrations and showed a maximum at around 100 mM, above which it decreased hyperbolically. 3. The ratio between the rate of transfer, vT, and that of hydrolysis of maltose, vW, vT/vW increased with the concentration of φG, showing that the hydrolysis of M is inhibited by φG. 4. Methyl alcohol was found to serve as an acceptor, judging from the formation of methyl α-glucoside detected by TLC. 5. The hydrolysis of M, and the transfer action of a glucosyl residue of M to φG and to M were explained by assuming essentially the same mechanism as that previously proposed for the hydrolysis of φM.

Disaccharide absorption by amphibian small intestine in vitro.

1. An account is given of the absorption of disaccharides by the small intestine of Rana temporaria, R. pipiens and Bufo vulgaris perfused in vitro through the vascular system. Maltase and trehalase activity are found in the intestine of all three species; very small amounts of sucrase are present in the intestine of R. pipiens but there is no evidence for the presence of lactase in any of the animals studied.2. During maltose absorption free glucose appears in the vascular effluent and in the intestinal lumen. Only very small quantities of disaccharide are found in the vascular effluent. The concentration of free glucose in the intestinal lumen during maltose absorption is not high enough to account for the rates of glucose transport observed. The rate of appearance of glucose in the vascular effluent is determined by the concentration of disaccharide in the luminal fluid, and hexose, free in solution in the lumen, is not an obligatory intermediate in the process of disaccharide absorption.3. For R. pipiens more than 90% of the maltase activity in the system is present in the intestinal wall and the rate of maltose hydrolysis by maltase, free in the intestinal lumen, is found to be inadequate to account for the rates of appearance of glucose observed to occur in the lumen and in the vascular effluent. It is not possible to wash away maltase activity from the intestinal wall.4. The kinetic properties of maltase and trehalase acting in situ are of the Michaelis-Menten type; the apparent K(m) is 2 mM for maltase, and 3 mM for trehalase.5. The relationship which exists between the rate of absorption of glucose and the concentration in the luminal fluid of either disaccharide or free glucose is of the Michaelis-Menten type. Expressed in molar units, the apparent K(m) for the glucose transport is about one fifth that of the disaccharidase. The maximum rate of glucose transport observed is less than the maximum rate of disaccharide hydrolysis. In R. pipiens equimolar concentrations in the intestinal lumen of the monomer free glucose, or of the dimer, maltose, yield approximately equal rates of transport of the free hexose.6. It is concluded that in the amphibian, either intestine disaccharide hydrolysis and glucose transport are functions of separate subcellular systems which spatially are very closely related, or that the hydrolysis and transport are different facets of the activity of a common system.

Hydrolysis of maltose by Taka-amylase A.

1. Maltose was found to be hydrolyzed at pH5.3 and 25°C by Taka-amylase A [EC 3.2.1.1] preparation which was crystallized three times and which was proved to be of single protein component by electrophoretic and ultracentrifugal analyses. 2. The Michaelis constant Km for the hydrolysis of maltose was found to be numerically equal to the competitive inhibitor constant Kt of maltose for the hydrolysis of p-nitrophenyl α-maltoside catalyzed by Taka-amylase A. 3. This Km value for maltose is one hundred times larger than the Km. value for the hydrolysis of maltose catalyzed by Taka-maltase [EC 3.2.1.20] or Taka-amylase B [EC 3.2.1.3] preparation. 4. A treatment of Taka-maltase at pH8.8 and 37°C for 2hr decreases its specific activity towards maltose, but the same treatment of the crystalline Taka-amylase A preparation causes no loss of specific activity towards maltose. On the other hand, a treatment with 1% HgCl2 solution at pH6.8 and room temperature for 24hr for crystalline Takaamylase A preparation decreases its specific activity towards maltose, but the same treatment for Taka-amylase B causes no loss of specific activity towards maltose. 5. From the results, it was concluded that the observed hydrolysis of maltose was caused by the action of Taka-amylase A itself and not by Taka-maltase or Taka-amylase B which might be contaminated in the crystalline preparation of Taka-amylase A. 6. The Michaelis constant Km for the hydrolysis of maltose catalyzed by Taka-amylase A was determined to be 9.6×10−2M and its breakdown rate constant k+2 of ES complex was determined to be 0.019 sec−1.

Role of a cell-wall glucan-degrading enzyme in mating of Schizophyllum commune.

Mycelial enzyme extracts of Schizophyllum commune were prepared during vegetative growth matings leading to common-A and common-B heterokaryons and the dikaryon, and were examined for hydrolytic activity against an alkaliinsoluble cell-wall glucan (R-glucan) isolated from this mushroom. In extracts from several individual homokaryotic mycelia the R-glucanase activity was low and did not increase when the cultures exhausted glucose in the medium. In common-A matings, a 30-fold increase in specific activity of intracellular R-glucanase was found even in the presence of glucose in the broth. An increase of this magnitude was not observed in the common-B mating nor in the fully compatible cross leading to the dikaryon. Extracts of the dikaryon did show elevated R-glucanase activity after exogenous glucose disappearance and subsequent fruiting. In none of these situations was an enzyme activity detected towards an alkali-soluble cell-wall glucan (S-glucan) prepared from S. commune. Changes in R-glucanase were not parallelled by identical changes in laminarinase, pustulanase, cellobiase, and p-nitrophenyl-beta-d-glucosidase, but comparable increases in specific activities were found for hydrolysis of glycogen and maltose. After interaction of the various mycelia in mating combinations, the S-glucan/R-glucan ratio of the cell wall of the dikaryon was found to be similar to that of the homokaryons, but increased in the common-B interaction and was elevated almost threefold in the common-A heterokaryon.

A continuous fermentation technique for studying the kinetics of sugar uptake by baker's yeast.

1. Solutions of glucose, maltose or other sugars were pumped at controlled rates into a yeast suspension and the extracellular sugar concentration was determined. The technique was especially suitable for studying the kinetics of fermentation at low rates of sugar utilization. At high rates the fermentation system was unstable. 2. Glucose fermentation was fitted by a model in which a diffusion barrier with first-order kinetics is interposed between the environment and the site of hexokinase. 3. Aerobic conditions affected the fermentation enzyme system but not the diffusion mechanism. 4. The kinetics of maltose fermentation at low rates approximated to those of the hydrolysis of maltose by the enzyme maltase, as studied in suspensions of broken cells.

DISACCHARIDASE DEFICIENCY IN INFANCY AND CHILDHOOD

CARBOHYDRATES provide a substantial proportion of the total caloric content of our diet. The dietary carbohydrates of quantitative importance are the polysaccharide starch and the disaccharides sucrose and lactose. Hydrolysis of each of these to monosaccharide form is necessary before significant absorption can occur. The principal product of starch digestion by amylases of the salivary glands and pancreas is maltose, a disaccharide in which two glucose units are joined by 1-4 α linkages. The other products are oligosaccharides including the disaccharide isomaltose, in which 1-6 α linkages are present. Further hydrolysis of maltose and oligo 1-6 glucosides, produced from starch, and the hydrolysis of sucrose and lactose ingested as such, is dependent on enzymes of the small intestinal mucosa, the disaccharidases. Figure 1 summarizes the sequence of digestion of the common dietary carbohydrates. For many years, clinicians have recognized that diarrhea in some patients is related to the ingestion of certain forms of carbohydrate. Recent years have seen an increasing interest in the detection of such patients, and the application of new techniques to their investigation. The development of instruments for intraluminal biopsy of the small intestinal mucosa was a notable advance, since it allowed specimens of this tissue to be obtained and some of its digestive capacities, including those concerned with disaccharide digestion, to be studied directly. By these means, it has been established that deficiency of one or more of the intestinal disaccharidases is often the basis of disaccharide intolerance. The terms "disaccharide intolerance" and "disaccharidase deficiency" are frequently used in this article, but have rather different meanings, so that a preliminary discussion of the sense in which each is used seems warranted.

Post-ruminal Degradation and Absorption of Carbohydrate by the Mature Ruminant

Sheep that had been fed a high carbohydrate ration were killed 3, 6 or 24 hours post-feeding to determine the relative amount and form of carbohydrate reaching the abomasum. The carbohydrate concentrations in abomasum contents, 3 hours post-feeding, was equal to 55% of the ration concentration. More total carbohydrate, due to more starch, was observed in the abomasum 3 hours post-feeding than 6 or 24 hours post-feeding. Starch and maltose were hydrolyzed and absorbed as glucose from the small intestine. The rate of hydrolysis of maltose and absorption of glucose was appreciably faster than the rate of blood glucose clearance, whereas the rate of starch hydrolysis was only slightly greater than the rate of blood glucose clearance. The latter difference was detectable only when a large amount of soluble starch was administered into the abomasum of sheep fed a high concentrate diet.