Neutral maltase of human granulocytes: localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.     

Purification and properties of neutral maltase from human granulocytes.

A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1----2), alpha (1----3) and alpha (1----4) glucosidic linkages. The highest activities were observed for alpha (1----4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.     

Column chromatography of human small-intestinal maltase, isomaltase and invertase activities

1. The maltase, isomaltase and invertase (sucrase) activities of solubilized mucosal preparations from human jejunum and ileum were studied with column chromatography on anion-exchange (diethylaminoethyl- and triethylaminoethyl-)cellulose and Sephadex G-200 gel. 2. On ion-exchange cellulose columns both kinds of enzyme preparations yielded two major disaccharidase peaks. The first peak contained maltase Ia (=isomaltase) and maltase Ib (=invertase). The second peak contained maltase II and maltase III. 3. On Sephadex G-200 gel columns jejunal preparations yielded the corresponding peaks as on ion-exchange columns, but the peaks appeared in the reverse order in the effluent. The ileal preparation studied yielded a single peak on gel columns, containing all the activities studied and eluted with the `void volume'. 4. Precipitation with ethanol did not affect the behaviour of the enzymes during ion-exchange chromatography. When gel filtration was performed after ethanol precipitation of the enzymes, however, two peaks were obtained also with the ileal preparation, and subfractionation of the invertase was obtained with both kinds of preparations. 5. The second peak from ion-exchange chromatograms, containing maltase II and maltase III, on concentration was found to have very weak isomaltase activity, probably exerted by these enzymes as such. This activity accounts for only about 1% of the total isomaltase activity of the mucosa. 6. The results support the concept of the specificity of the human small-intestinal disaccharidases previously described after heat-inactivation experiments. The subfractionation of the invertase that under certain conditions is seen on Sephadex G-200 columns appears most likely to be an artifact. Consequently the nomenclature for the human maltose-, isomaltose- and sucrose-splitting enzymes proposed by another research group after gel-filtration chromatography studies should be abandoned. It seems more logical to keep the nomenclature based on heat inactivation [maltase Ia (=isomaltase), maltase Ib (=invertase or sucrase), maltase II and maltase III] until increased knowledge about the specificity and structure of these enzymes makes possible a more rational nomenclature.     

Substrate specificity and kinetic properties of neutral maltase of human granulocytes

A neutral maltase immunologically similar to this of kidney exist in human granulocytes. We have studied some kinetic properties of this enzyme on a microsomal fraction of granulocytes. Its optimal pH is very closed of 6.8 and this enzyme, highly specific for maltose, hydrolysis very weakly the nigeriosis. Maltotriose, maltotetraose and maltopentanose are inhibitors of this enzyme, which is not inhibited by all disaccharides studied.     

Synthesis of maltotriose by maltase purified from rabbit kidney.

To clarify the enzyme participates in maltotriose synthesis, we purified maltase from rabbit kidney using 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) affinity column chromatography. The purified enzyme possessed specific activity of 33.7 mumol/mg/min and estimated molecular weight of 350,000 dalton, as judged by SDS-polyacrylamide gel electrophoresis, comparable with those reported from rat kidney. Moreover this enzyme possessed not only maltase (maltose----glucose) but also amylomaltase (maltose----maltotriose) activity, and both activities were inhibited by Tris in a dose-dependent manner with similar IC50 values. From these results, we concluded that maltotriose was synthesized by maltase in vitro and that kidney maltase may participate in sugar metabolism in vivo.     

Comparisons of maltase activities in kidney brush border membranes from normal, diabetic, glucose-infused and maltose-infused rabbits

The specific activities of membrane-bound maltase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) in isolated brush border membranes (BBMs) of alloxan-induced diabetic, glucose-infused and maltose-infused rabbits were 30%, 140% and 160%, respectively, of those of control rabbits. Differences in the relative activities of trehalase (EC 3.2.1.28), another disaccharidase, in these groups were similar but less marked. However, the activities of two other marker enzymes of the brush border, alkaline-phosphatase and gamma-glutamyl transpeptidase, were similar in the 4 groups of rabbits. The decreases in the activities of the two disaccharidases were due to changes in the Vmax values of the enzymes without change in their Km values for maltose and trehalose. The maltase activities in the 4 groups showed similar dependences on Tris-HCl, KCl and NaCl. The electrophoretic profiles of the BBMs of the 4 groups on SDS-polyacrylamide gel showed slight differences. From these results, we conclude that diabetes, glucose infusion and maltose infusion probably change the concentrations of active enzymes in the BBM of the kidney in rabbits.     

Val216 decides the substrate specificity of alpha-glucosidase in Saccharomyces cerevisiae.

Differences in the substrate specificity of alpha-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing alpha-1,4- and alpha-1,6-glucosidic linkages, two alpha-glucosidases, maltase and isomaltase, from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was 72.1% sequence identity with 165 amino acid alterations between the two alpha-glucosidases. These two alpha-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified alpha-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the consensus region II, which is one of the four regions conserved in family 13 (alpha-amylase family), is replaced with the maltase type, the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two alpha-glucosidases. Thus, we modified Val216, Gly217, and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity, indicating that Val216 is an important residue discriminating the alpha-1,4- and 1,6-glucosidic linkages of substrates.     

Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase-glucoamylase identical with membrane-bound maltase-glucoamylase in molecular weight, heat-sensitivity, substrate specificity, K(m) for maltose and K(i) for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase-glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its K(m) for maltose was 1.5mm. It was inhibited by turanose (K(i)=7.5mm) and Tris (K(i)=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50 degrees C for 10min. The acid maltase closely resembled beta-glucuronidase and acid beta-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.     

Selectivity of 3'-O-methylponkoranol for inhibition of N- and C-terminal maltase glucoamylase and sucrase isomaltase, potential therapeutics for digestive disorders or their sequelae

Human maltase glucoamylase (MGAM) and sucrase isomaltase (SI) are two human intestinal glucosidases responsible for the final step of starch hydrolysis. MGAM and SI are anchored to the small intestinal brush border epithelial cells and contain two catalytic N-terminal and C-terminal subunits. In this study, we report the inhibition profile of 3'-O-methylponkoranol for the individual recombinant N and C terminal enzymes and compare the inhibitory activities of this compound with de-O-sulfonated ponkoranol. We show that 3'-O-methylponkoranol inhibits the different subunits to different extents, with extraordinary selectivity for C-terminal SI (K(i)=7±2nM). The enzymes themselves could serve as therapeutic targets for the treatment of digestive disorders or their sequelae.     

Pyelonephritis alters the reabsorption of nutrients and brush border membrane enzymes of rat kidney

The uptake of nutrients and activities of membrane enzymes in the kidney were investigated using renal brush border membrane (BBM) vesicles in acute pyelonephritis in rats. A significant decrease (P less than 0.001) in the uptake of D-glucose and L-phenylalanine was observed in both the unobstructed right and obstructed left kidney, while there was a significant increase (P less than 0.001) in the uptake of L-alanine in the left kidney of pyelonephritic rats, demonstrating disturbances in the reabsorption of the glucose and aminoacids in the kidneys. Vmax of alkaline phosphatase, leucine-amino-peptidase and maltase was found to be decreased in the left kidney, suggesting that there was a reduction in the active enzyme molecule number. Km of alkaline phosphatase and leucine-aminopeptidase remained unchanged, while km of maltase decreased in both the right and left kidneys. An increase in the Vmax of alkaline phosphatase and leucine-aminopeptidase and substrate affinity of the maltase in the right kidney demonstrated a compensatory phenomenon for the malfunctioning of the left kidney. This is the first report demonstrating alterations in reabsorption of nutrients and BBM enzymes in experimental pyelonephritis.     

Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeastHansenula polymorpha: Isolation and study of regulatory mutants

Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeastHansenula polymorpha. Maltase was shown to be responsible for the growth ofH. polymorpha not only on maltose, but also on sucrose. The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (pNPG) <sucrose <maltose. Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained fromH. polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-d-glucose. One of the isolated mutants, L63, was studied in more detail. Mutant L63 was recessive and monogenic and it was not deficient in hexokinase. Its analysis revealed thatH. polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.     

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes.     

Substrate specificity of three prostaglandin dehydrogenases

Studies on the substrate specificity, kcat/Km, and effect of inhibitors on the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase (9-ketoprostaglandin reductase) indicate that it is very similar to a human brain carbonyl reductase which also possesses 9-ketoprostaglandin reductase activity. These observations led to a comparison of three apparently homogeneous 15-hydroxyprostaglandin dehydrogenases with varying amounts of 9-ketoprostaglandin reductase activity: an NAD- and an NADP-linked enzyme from human placenta and an NADP-linked enzyme from rabbit kidney. All three enzymes are carbonyl reductases for certain non-prostaglandin compounds. The placental NAD-linked enzyme, which has no 9-ketoprostaglandin reductase activity, is the most specific of the three. Although it has carbonyl reductase activity, a comparison of the Km and kcat/Km for prostaglandin and non-prostaglandin substrates of this enzyme suggests that its most likely function is as a 15-hydroxyprostaglandin dehydrogenase. The results of similar comparisons imply that the other two enzymes may function as less specific carbonyl reductases.     

Neutral maltase/glucoamylase from rabbit renal cortex.

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.     

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.     

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.