Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.     

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

A specific acid alpha-glucosidase in lamellar bodies of the human lung

In the present investigation, we have demonstrated that three lysosomal-type hydrolases, alpha-glucosidase, alpha-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid alpha-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal alpha-glucosidase from human placenta, two alpha-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of alpha-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all alpha-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the alpha-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body alpha-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid alpha-glucosidases: one similar to the lung lysosomal alpha-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific alpha-glucosidase should prove useful as a lamellar body-specific marker enzyme.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Interaction of hepatic asialoglycoprotein receptor with asialoorosomucoid and galactolyzed lysosomal alpha-glucosidase

An asialoglycoprotein receptor was isolated from murine liver and purified more than 1600-fold using 2-fold affinity chromatography on asialoorosomucoid-Sepharose. The purified receptor did not interact with 125I-orosomucoid, but bound to 125I-asialoorosomucoid. The binding of the receptor to asialoorosomucoid was saturable. The dissociation constant of the receptor-asialoorosomucoid complex was 0.4 X 10(-9) M. The molecular mass of the receptor, as determined with the use of specific antibodies by the immunoblotting method, was 43 kDa. High concentrations of unlabeled asialoorosomucoid and of n-aminophenyl-beta-D-galactosyl derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase from human liver inhibited the binding of the receptor to 125I-asialoorosomucoid almost completely. The binding of the receptor to 125I-galactolyzed alpha-glucosidase was pH-dependent, with the pH optimum at 8.0-9.0. It was shown that, as in the case of 125I-asialoorosomucoid, the binding of the 125I-galactosyl derivative of alpha-glucosidase occurred in the presence of Ca2+ and was inhibited by N-acetylgalactosamine. Glycoproteins containing galactose as a terminal residue inhibited the interaction of the receptor with 125I-galactolyzed alpha-glucosidase. The possibility of directed transport of the galactolyzed alpha-glucosidase derivative into parenchymous liver cells using receptor-mediated endocytosis is discussed.     

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Further studies of the structure of human placental acid alpha-glucosidase

Acid alpha-glucosidase has been purified from human placenta to a specific activity of approximately 6800, (4-methylumbelliferyl-alpha-D-glucoside as a substrate) or 55,400 mumol g-1 min-1 (glycogen or maltose as substrate). The purified enzyme gives rise to multiple protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), i.e., a major doublet of 82K and 69K , a minor doublet of 25K and 21K , and a faint band of 100K. All of the molecular weight species stained as glycoproteins with an intensity apparently proportional to their protein content, and were present in enzyme from individuals homozygous for the allozyme alpha-Glu 1. Isoelectric focusing revealed only enzymatically active proteins which, when analysed by SDS-PAGE, gave rise to multiple molecular weight species. Chromatography of I125-labeled, purified enzyme on Bio-Gel P-100 revealed only a radiolabeled, high-molecular-weight species which corresponded with enzyme activity. These findings suggest that, in the native state, the mature enzyme exists as a high-molecular-weight species, which is dissociable in SDS to several low-molecular-weight species. These results are consistent with reports that a 100K primary product of translation is post-translationally modified to yield polypeptides of lower molecular weights, and that all of the molecular species are absent in cells genetically deficient for acid alpha-glucosidase. The possibility that the low-molecular-weight (20- 25K ) protein bands in SDS-gels corresponded to a previously reported low-molecular-weight species generated by treatment with guanidine-HCl was investigated. The I125-labeled, purified acid maltase was dissociated by guanidine into two equal peaks of approximately 64K and 28K molecular weight. Surprisingly, both peaks, when analyzed on SDS-gels, yielded identical and equally intensely staining bands of 64K molecular weight. These results suggest that the mature acid alpha-glucosidase is made up of polypeptides which are bonded in the native state by at least two different types of interaction, one type which is dissociable in SDS and one type which is dissociable in guanidine but not in SDS. The nature and possible function of the 25K polypeptide generated only by guanidine-HCl remains to be determined.     

Uptake and stability of human and bovine acid alpha-glucosidase in cultured fibroblasts and skeletal muscle cells from glycogenosis type II patients

Acid alpha-glucosidase (EC 3.2.1.20) was purified from human placenta and bovine testis by affinity chromatography using concanavalin A (conA) and Sephadex G 200. When added to the culture medium of human fibroblasts, the enzyme purified from bovine testis is taken up with a 200-fold higher efficiency than the enzyme from human placenta. Uptake of acid alpha-glucosidase from bovine testis is mediated by the mannose-6-phosphate receptor, whereas only a minor fraction of placental enzyme appears to be equipped with the mannose-6-phosphate recognition marker. Once internalized, both human and bovine acid alpha-glucosidase demonstrate a half-life of about 10 days in fibroblasts from control individuals and patients with different clinical forms of glycogenosis type II (Pompe's disease, acid alpha-glucosidase deficiency). Evidence is presented that the mannose-6-phosphate receptor is also present on the plasma membrane of the clonal myogenic skeletal muscle cell lines G8-1 and L6J1 (respectively from mouse and rat origin) and on cultured human skeletal muscle cells derived from a muscle biopsy. Addition of bovine testis acid alpha-glucosidase to skeletal muscle cell cultures from an adult patient with glycogenosis type II leads to complete correction of the enzyme deficiency.     

Use of immobilized antibodies in investigating acid alpha-glucosidase in urine in relation to Pompe's disease.

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.     

A thermostable acid alpha-glucosidase from Tetrahymena thermophila: purification and characterization

An acid alpha-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56 degrees C and showed resistance to thermal inactivation. The acid alpha-glucosidase appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid alpha-glucosidase.     

Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey

Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.     

alpha-Glucosidase, a membrane-bound enzyme of alpha-glucan metabolism in Bacillus amyloliquefaciens. Purification and partial characterization.

The organism Bacillus amyloliquefaciens is capable of producing alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) extracellurlarly and a membrane-bound, intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). The amounts of alpha-glucosidase in cells of B. amyloliquefaciens grown on amylaceous polysaccharides were significantly higher then in cells grown on non-carbohydrate carbon sources. alpha-Glucosidase was exclusively found associated with membranes from ruptured spheroplasts by subcellular fractionation and solubilization studies. Salt solutions and chelating agents alone did not dislodge alpha-glucosidase from membranes, but in combination with detergents were most effective in solubilizing active enzyme (0.1% sodium cholate (pH 8.0)/0.4 M sodium chloride). Purified alpha-glucosidase very rapidly hydrolized p-nitrophenyl alpha-D-glucopyranoside and sucrose. Maltose, maltotriose, isomaltose and isomaltotriose were hydrolized at slower rates, whereas beta-glucosides and polymeric alpha-glucans were not attacked. Other properties of the purified enzyme were as follows: Temperature optimum for catalysis = 39 +/- 1 degrees C; pH optimum = 6.8; molecular weight = 27,000 +/- 1000. alpha-Glucosidase is proposed to function in the endogenous metabolism of alpha-glucans provided extracellularly as carbon sources for growth of B. amyloliquefaciens.     

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.     

Solubilization of membrane-bound acid alpha-glucosidase by proteolysis

The membrane-bound acid alpha-glucosidase was purified partially (400-fold) from human placenta by solubilization with trypsin, concanavalin A-Sepharose chromatography, Ultrogel AcA-34 gel filtration, and Sephadex G-100 affinity chromatography. Two molecular forms of the enzyme were found in the final preparation of the purified enzyme. They were identical in molecular weight with a precursor (110 kDa) and an early intermediate form (105 kDa) of this enzyme. Also direct incubation of the membrane fraction without trypsin resulted in a release mainly of the 105 kDa form, which was inhibited by N-ethylmaleimide, but not by leupeptin, pepstatin or phenylmethylsulfonylfluoride. It was concluded that the precursor of acid alpha-glucosidase is an intrinsic membrane protein, which is transported into lysosomes after solubilization by proteolysis.     

On the origin of alpha-glucosidase in human urine

The recent findings that alpha-glucosidase from human kidney was identical with one component (F1) of the alpha-glucosidases found in human urine suggested the idea that this enzyme might originate in the kidneys. The present study was performed to test this idea by immunological methods. Urine alpha-glucosidase F1 was isolated in the electrophoretically homogeneous state, and the antibody prepared in rabbits was purified by affinity chromatography after the antisera were fractionally precipitated with ammonium sulfate and chromatographed on diethylamino ethyl (DEAE)-cellulose. The staining of human kidney tissue sections was performed by the indirect method, using alpha-glucosidase F1 antibody and fluorescein-conjugated anti-rabbit immunoglobulin goat sera. The proximal convoluted portion (proximal tubules) with brush border and Henle's loops (late proximal) were stained clearly. Preincubation of intact antibody with purified antigen prevented specific staining of the proximal convoluted portion and Henle's loops. In contrast, all other tissues of kidney were stained less positively or negatively. These results indicate that alpha-glucosidase F1 originates in the kidney, and that glucosidase is specifically localized in the proximal convoluted portion and Henle's loops.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.     

Neutral alpha-glucosidase in granule fractions from guinea pig polymorphonuclear leukocytes: enzymic characterization and comparative studies with monoclonal antibodies

Neutral alpha-glucosidase was partially purified from granular fractions isolated from guinea pig polymorphonuclear leukocytes (PMNL). The native enzyme had a high molecular weight, about 417,000, with a subunit of 43,000. The purified enzyme hydrolysed 4-methylumbelliferyl alpha-glucoside and maltose, but not isomaltose, trehalose, and glycogen. The enzyme was strongly inhibited by bromoconduritol and castanospermine, but only slightly by turanose. Monoclonal antibodies which can bind specifically to the enzyme were prepared by immunizing mice with the partially purified enzyme. Hybridomas producing the monoclonal antibodies were selected by an enzyme-linked immunosorbent assay. The seven monoclonal antibodies were found to react with the enzyme from PMNL, but not with the glycoprotein-processing alpha-glucosidase isolated from liver microsomes nor with the macrophage enzyme. The results indicated that PMNL contain a particulate neutral alpha-glucosidase enzymologically and immunologically distinct from other alpha-glucosidases.