Isolation and Purification of an alpha-Mannosidase from Coleoptiles of Avena sativa

An alpha-mannosidase has been purified from the coleoptiles of Avena sativa L. var. Segrehavre. The enzyme, which is tightly associated with the cell wall, was solubilized with 3 m LiCl. The purification involves precipitation with (NH(4))(2)SO(4), gel filtration, ion exchange chromatography, and isoelectric focusing. The enzyme appears homogeneous when chromatographed on disc gels and on isoelectric focusing gels. The enzyme runs as a single protein of constant specific activity when chromatographed on Sephadex G-200. The estimated molecular weight of the enzyme is 630,000. The enzyme appears to have no metal ion cofactor requirement and is insensitive to p-chloromercuribenzoate. The pH optimum for the enzyme with p-nitrophenyl-alpha-d-mannoside as the substrate is 4.5 and the K(m) is 3.2 mm. The enzyme may have some carbohydrate associated with it as indicated by a positive periodate-Schiff reaction on disc gels.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.     

Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice

AIMS: To purify and characterize an extracellular alpha-glucosidase from Trichoderma viride capable of inactivating a host-specific phytotoxin, designated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia solani Kühn. METHODS AND RESULTS: The host-specific RS toxin was purified from both culture filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, were carried out. The antifungal activity of the purified high molecular weight protein (110 kDa) was studied against RS toxin as well as on the sclerotial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that the protein was an alpha-glucosidase. The 110-kDa protein was highly specific to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l-1 when p-nitrophenyl alpha-D-glucopyranoside was used as the substrate. The isoelectric point of the protein was 5.2. N-terminal sequencing of the alpha-glucosidase protein showed that its amino acid sequence showed no homology with other known alpha-glucosidases. CONCLUSION: This appears to be the first report of the purification and characterization of an alpha-glucosidase capable of inactivating a host-specific toxin of fungal origin. The alpha-glucosidase is specific to RS toxin and is different from the known alpha-glucosidases. SIGNIFICANCE AND IMPACT OF THE STUDY: As RS toxin could be inactivated by the microbial alpha-glucosidase enzyme, isolation of the gene that codes for the enzyme from T. viride and transfer of the gene to rice plants would lead to enhanced resistance against sheath blight pathogen by inactivation of RS toxin.     

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.     

Purification and properties of UDP-glucose galactosylhydroxylysine collagen glucosyltransferase (EC 2.4.1.?) from bovine arterial tissue.

The glucosyltransferase (UDP-glucose galactosylhydroxylsine collagen glucosyltransferase, EC 2.4.1.?.) was purified 50-fold from calf arterial tissue by ammonium sulfate precipitation, gel filtration and electrofocusing. The purified enzyme has a molecular weight of 72 000 and a requirement for Mn2. It resolves into two activity peaks when submitted to electrofocusing (isoelectric point at pH 4.2 and 8.1) or disc electrophoresis and exhibits a double pH optimum (pH 8.3 and 9.9). The enzyme was found to transfer glucose from UDP-glucose to the denatured forms of citrate-soluble calf skin collagen (I), the alphal chain (II) and the beta12 component (III) derived from it, and of an acetic-acid-souble collagen preparation (IV) obtained from alkali-treated calf arterial tissue. The Km values for the substrates were 1.67 X 10(-4) (I), 6.3 X 10(-4) (II), 3.3 X 10(-4) (III) and 2.8 X 10(-4) mol/l (IV), indicating that the enzyme has the greatest affinity for the calf skin collagen. The glucose transferred to hydroxylysine-linked galactose residues may be released subsequently by the action of a specific alpha-glucosidase purified from bovine spleen. The results support the assumtion that the glucosylation step in the course of the (pro-)-collagen biosynthesis depends on special structural features of the substrate and may be controlled by a specific alpha-glucosidase.     

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.     

One-step purification and properties of catalase from leaves of Zantedeschia aethiopica

Catalase (E.C 1.11.1.6) was purified from leaves of Zandedeschia aethiopica to apparent homogeneity by a one-step hydrophobic interaction chromatography on a phenyl Sepharose CL-4B column. The purified enzyme preparation was obtained with a final recovery of enzyme activity of about 61% and a specific activity of 146 U/mg protein. The purified enzyme ran as a single protein band when analyzed both by native PAGE and SDS-PAGE corresponding to an Mr of 220,000 Da, which consists of 4 subunits with identical Mr of 54,000 Da. The pI of purified enzyme was found to be 5.2 by isoelectric focusing on ultrathin polyacrylamide gels. The purified catalase has an optimum temperature of activity at 40 degrees C, whereas it is stable between 0 degrees and 50 degrees C. As regards pH, the enzyme has an optimum activity at pH 7.0 and it is stable in the range pH 6-8. The absorption spectrum of the purified enzyme exhibited 2 peaks at 280 nm and 405 nm.     

Isoelectric focusing studies on the beta-fructofuranosidases and alpha-glucosidases of Streptococcus mitis.

Extracts of Streptococcus mitis ATCC 903 were analysed for beta-fructofuranosidase and alpha-glucosidase activities by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures. Three bands of activity were visualized in the gels after incubation with sucrose (pI 4.05, 4.25 and 4.85) and three other bands after incubation with p-nitrophenyl alpha-D-glucopyranoside (pI 3.90, 4.45 and 4.65). The enzymes responsible for the reaction with sucrose were identified as beta-fructofuranosidases (EC 3.2.1.26) for the following reasons: identical enzyme bands were visualized in the gels after incubation with raffinose; no enzyme bands appeared in the gel after incubation with the alpha-glucosides maltose, turanose, trehalose and melezitose; and the soluble fraction hydrolysed sucrose to equimolar amounts of glucose and fructose.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Purification and properties of peroxidase from Pinus pinaster needles

Peroxidase (Ec 1.11.1.7) was purified from needles of Pinus pinaster to apparent homogeneity by DE-52 cellulose chromatography with a final recovery of enzyme activity of about 85%. The purified enzyme (A402/A275 = 1.05) had a specific activity of about 948 U/mg of protein and ran as a single protein band both on SDS-PAGE and native PAGE with Mr of 37,000 and 151,000, respectively. Both native PAGE and isoelectric focusing gels of the purified enzyme were stained for activity which coincided with the protein band. The pI of the purified enzyme was found to be 3.2 by isoelectric focusing on an ultrathin polyacrylamide gel. The enzyme has an optimum pH of activity of 5.0 and temperature optimum of 30 degrees C. Stability studies of the enzyme as a function of pH and temperature suggest that it is most stable at pH 5.0 and 0-40 degrees C, respectively.     

Purification and characterization of a new type of alpha-glucosidase from Paecilomyces lilacinus that has transglucosylation activity to produce alpha-1,3- and alpha-1,2-linked oligosaccharides

A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.     

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.     

Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 3. Purification and physico-chemical characterization of an exo-1,4-beta-glucanase.

An exo-1,4-beta-glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico-chemical properties. The purification has been carried out in a five-step procedure comprising chromatography on DEAE-Sephadex, gel filtration on polyacrylamide P-150, activation on a Dowex 2-X8 anion exchanger, chromatography on Concanavalin A-Sepharose and chromatography on SP-Sephadex. The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo-1,4-beta-glucanase activity 7%. After purification, the enzyme showed no viscosity-decreasing activity towards carboxymethyl-cellulose solutions. The exo-1,4-beta-glucanase was isoelectric at pH 4.3 (4 degrees C). A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.     

alpha-L-arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall.

An alpha-L- arabinofuranosidase has been purified from the extracellular broth of cultures of Ruminococcus albus 8. The purification procedure utilized gel filtration, (NH4)2SO4 precipitation, and isoelectric focusing. The purified enzyme appeared to be homogeneous when chromatographed on disc and analytical isoelectric focusing gels. The estimated molecular weight of the native protein was 305,000 to 310,000. Sodium dodecyl sulfate-gel electrophoresis analysis suggested that the native protein is a tetramer composed of 75,000-molecular-weight subunits. The enzyme appeared to have no metal cofactor requirement but was sensitive to several sulfhydryl reagents. The pH optimum with p-nitrophenyl-alpha-L-arabinofuranoside as the substrate was 6.9 and the Km was 1.3 mM. Several lines of evidence indicated that the enzyme is a glycoprotein. When assayed against alfalfa cell wall material, the enzyme hydrolyzed only small amounts of arabinose from the substrate. When assayed together with hemicellulolytic or pectinolytic enzymes against the same substrate, the arabinosidase significantly enhanced the hydrolytic action of the glycanases .     

Purification to homogeneity and some properties of L-phenylalanine ammonia-lyase of irradiated mustard (Sinapis alba L.) cotyledons.

1. Lyase (L-Phenylalanine ammonia-lyase, EC 4.3.1.5) from far-red light-irradiated mustard cotyledons was purified to a single protein using ammonium sulphate fractionation, column chromatography on L-phenylalanyl-Sepharose 4B and on Sephadex G-200, isoelectric focusing and polyacryalmide gel electrophoresis. 2. The enzyme constituted 0.01% of total cellular protein, did not catalyse the deamination of L-tyrosine, had a pH optimum of pH 8.6 and an isoelectric point of pH 5.6. 3. The sedimentation coefficient was estimated as 11.3 S, the Stokes' radius 4.25 nm, and the molecular weight 240 000 +/- 9000 (S.E.). 4. Electrophoresis on denaturing polyacrylamide gels gave a single stained protein band corresponding to a subunit molecular weight of 55 000 indicating a tetrameric structure of equal (or near-equal) size subunits. 5. Maximum velocity (V) for the purified lyase at 25 degrees C was 3.83--4.10 nkat. 1(-1) enzyme and Km value 0.151--0.154 mM. Negative cooperativity (Hill coefficient, n = 1.08) was not detected over the substrate concentration range tested. 6. A putative non-diffusible inhibitor isolated from dark-grown gherkin hypocotyls inhibited the homogeneously purified mustard lyase.     

Extraction of acid alpha-glucosidase from human spleen]

An efficient method for isolation of acid alpha-glucosidase from human spleen is developed. The method involves chromatography of the enzyme on rho-aminophenyl-alpha-D-glucopyranoside covalently bound to CH-Sepharose 4B, with subsequent gel filtration on Sephadex G-200. The enzyme was homogeneous by the polyacrylamide gel electrophoresis data; it was purified about 1500-fold, as compared with the crude extract (the total yield 12.5%). Besides acid alpha-glucosidase, the preparations of alpha-L-fucosidase, alpha-D-galactosidase and beta-N-acetylglucosaminidase were isolated and purified 200-, 130- and 280-fold, respectively. The nature of interaction between acid alpha-glucosidase and immobilized rho-aminophenyl-alpha-glucopyranoside is discussed.     

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Caproyl esterase from rat testis: Purification and action on cumulus cells

Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein. The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate. The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.