Partial Purification and Characterization of Naegleria fowleriβ-Glucosidase1

Naegleria fowleri cells, grown axenically, contain high levels of β-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-β-D-glucopyranoside (4MUGlc) (K_m, 0.9 mM), octyl-β-D-glucoside (K_m, 0.17 mM), and p-nitrophenyl-β-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the β-glucosidase activity appears in the supernatant fraction. The β-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble β-D-galactosidase activity in the Naegleria extract copurifies with the β-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (K_i, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleriaβ-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 Å, and a sedimentation coefficient of 4.2S. The β-glucosidase is not inhibited by conduritol β-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol β-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the β-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

The occurrence of two immunologically distinguishable beta-glucocerebrosidases in human spleen

The beta-glucosidase activity in spleen from control subjects and patients with different clinical phenotypes of Gaucher's disease was characterized. The occurrence of a soluble non-specific beta-glucosidase with a neutral pH optimum and two membrane-associated beta-glucocerebrosidases with an acid pH optimum was demonstrated. The two beta-glucocerebrosidases can be distinguished on the basis of their ability to react with anti-(placental beta-glucocerebrosidase) antibodies bound to protein-A--Sepharose 4B beads. One of the splenic beta-glucocerebrosidases (form I) is precipitated by the immobilized antibodies and the other (form II) is not. The two forms also differ in binding affinity to concanavalin A, degree of stimulation of enzymic activity by taurocholate and isoelectric point. In contrast, the Km values of the two beta-glucocerebrosidases for natural and artificial substrates are similar and both are inhibited by conduritol B-epoxide. In spleen from three patients with type 1, one patient with type 2 and one patient with type 3 Gaucher's disease form I beta-glucocerebrosidase was found to be clearly deficient, whereas the activity of form II was 25-50% of that in control spleen. The non-specific, neutral beta-glucosidase was not deficient in these Gaucher spleens. The distinct biochemical and immunological properties of non-specific beta-glucosidase and the fact that normal levels of the enzyme are present in patients with Gaucher's disease indicate, in confirmation of previous reports, that non-specific beta-glucosidase is not related to beta-glucocerebrosidase.     

Modulation of renal epithelial cell growth by glucosylceramide. Association with protein kinase C, sphingosine, and diacylglycerol.

Two independent approaches were employed to explore the potential role of endogenous glucosylceramide or a closely related glucosphingolipid in mediating the cellular proliferation of Madin-Darby canine kidney cells. First, cultured cells were depleted of glucosphingolipids by exposure to a glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. This agent markedly inhibited cell growth and DNA synthesis in a time- and concentration-dependent manner. Second, cells were grown in the presence of conduritol B epoxide, an inhibitor of glucosylceramide beta-D-glucosidase. Exposure of cells to this inhibitor resulted in the time-dependent accumulation of glucosylceramide with a corresponding increase in cellular proliferation. Alterations in protein kinase C activity were evaluated as a potential mechanism for these effects on growth. Both membrane- and cytosol-associated protein kinase C (PKC) activity declined under conditions of glucosylceramide synthase inhibition and increased under conditions of beta-glucosidase inhibition. The changes in PKC activity were evident after DEAE-cellulose purification. Diacylglycerol levels increased in response to both glucosylceramide synthase and beta-glucosidase inhibition. Ceramide and sphingosine levels changed only in the presence of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, increasing due to lack of conversion to glucosylceramide. However, the elevation in endogenous sphingosine was probably insufficient to account for the decrease in PKC, considering the high level of diacylglycerol in the cells. These data demonstrate an association between glucosylceramide levels, PKC activity, and cell growth.     

Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri

Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta-galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

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%).     

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.     

Purification and properties of an induced beta-D-glucosidase from stachybotrys atra.

We have purified an induced beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra to apparent homogeneity. The induced enzyme was clearly different from the constitutive beta-D-glucosidase. The molecular weight was 65 500-69 000, the pH optimum was at 6.7 and the isoelectric point at 4.8. Carbohydrate content (related to glucose) was 14.4%. The enzyme showed beta-D-glucosidase, beta-D-xylosidase and beta-D-thioglucosidase activity. These three activities sppear to be due to the same enzyme. The enzyme was strongly inhibited by D-glucono-(1 leads to 5)-lactone and nojirimycin and was very sensitive to sulfhydryl reagents.     

The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.     

Heterogeneity in human acid beta-glucosidase revealed by cellulose-acetate electrophoresis

Cellulose-acetate gel electrophoresis, a technique commonly used for the separation of human acid hydrolases, was applied to study heterogeneity in acid beta-glucosidase (EC 3.2.1.45). With this technique, three forms of beta-glucosidase were distinguishable in extracts of several tissues. The most anodic beta-glucosidase activity (band 3) represents the broad-specificity beta-glucosidase that is not deficient in Gaucher disease and is not inhibited by conduritol B-epoxide (CBE). The beta-glucosidase activity was deficient in Gaucher disease. A third beta-glucosidase activity with an intermediate mobility (band 2) was also inhibited by CBE and deficient in Gaucher disease. Band 1 and band 2 beta-glucosidase thus represent different forms of glucocerebrosidase. By adding phosphatidylserine and sphingolipid activator protein (SAP-2), monomeric glucocerebrosidase could be completely converted into a form that comigrated with band 2 beta-glucosidase of tissue extracts. The addition of phosphatidylserine only also resulted in a changed mobility of the monomeric enzyme, but the migration in this case differed from that of band 2 beta-glucosidase of tissue extracts. The electrophoretic profile of beta-glucosidase activity of tissue extracts changed upon ethanol/chloroform extraction: the two glucocerebrosidase forms were converted into a band with a mobility identical to that of band 1 beta-glucosidase. Our findings indicate that the interaction of glucocerebrosidase with phospholipid and SAP-2 has major effects on the mobility of the enzyme in the cellulose-acetate gel electrophoresis system. The findings with the cellulose-acetate gel electrophoretic system are discussed in relation to the heterogeneity in glucocerebrosidase observed with sucrose density gradient analysis, immunochemical methods and isoelectric focussing studies.     

beta-Fucosidase, beta-glucosidase and beta-galactosidase activities associated in bovine liver

1. beta-D-Fucosidase, beta-D-glucosidase and beta-D-galactosidase activities from bovine liver are associated in a single peak in isoelectric focusing. The isoelectric point is 4.35 for all these activities, suggesting that they are catalyzed by the same enzyme. 2. This enzyme shows the optimal pH in the range 4.5-6.5 for all the above mentioned activities. 3. The Km and Vmax are 0.26 mM and 31 mU mg-1, 0.10 mM and 24 mU mg-1, and 0.30 mM and 20 mM mg-1 for the p-nitrophenyl-fucoside, -glucoside and -galactoside, respectively. The glucoside derivative is the best substrate, with a Vmax/Km value of 0.24 ml . mg-1 . min-1. 4. The Lineweaver-Burk profiles are convex upward in most cases, suggesting a substrate-activation model, and the presence of more than one binding site in the enzyme. 5. The Ki for all the activities were determined with D-fucose, glucose and galactose as inhibitors. D-Fucose is the strongest inhibitor. The inhibition is competitive in all cases.     

Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva).

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.     

Purification and Characterization of an Intracellular β-Glucosidase from the Methylotrophic Yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Conduritol aziridine: a new mechanism-based glucosidase inactivator

A new mechanism-based glucosidase inactivator, conduritol aziridine (1,2-dideoxy-1,2-epimino-myo-inositol), has been synthesised from myo-inositol. This aziridine inactivates both the beta-glucosidase from Alcaligenes faecalis and the alpha-glucosidase from yeast according to the expected pseudo-first order kinetics. Inactivation constants measured are Ki = 3.0mM, ki = 0.077 min-1 for the beta-glucosidase, and Ki = 9.5mM, ki = 0.39 min-1 for the alpha-glucosidase. Evidence for irreversible inactivation is provided by the lack of reactivation upon dilution of inactivated enzyme into buffer containing substrate.     

Comparison of the properties of a soluble form of glucocerebrosidase from human urine with those of the membrane-associated tissue enzyme

Human urine contains a soluble form of glucocerebrosidase, an enzyme associated with the lysosomal membrane in cells and tissues. Urinary glucocerebrosidase is identical to the enzyme extracted from tissues with respect to the following parameters: Km for natural and artificial substrates, inhibition by conduritol B-epoxide, and stimulation by taurocholate. The enzyme is greater than 90% precipitable by polyclonal anti-(placental glucocerebrosidase) antiserum. Upon isoelectric focussing of urinary glucocerebrosidase multiple peaks of activity were observed. Partial deglycosylation (removal of sialic acid, N-acetylglucosamine and galactose) of the urinary enzyme increased the isoelectric point to a value identical to that of the main form found after partial deglycosylation of the placental enzyme. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by immunoblotting, the immunopurified urinary enzyme shows the same molecular mass forms as the enzyme immunopurified from brain and kidney. In placenta the apparent molecular mass is somewhat higher but upon removal of sialic acid, N-acetylglucosamine and galactose the urinary and the placental enzyme show identical molecular masses of 57 kDa. We conclude that the enzymes extracted from urine and tissue are identical and that differences in apparent molecular mass and isoelectric point are probably due to heterogeneity in the oligosaccharide moieties of the molecules.     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins

The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.     

Hydrolysis of a naturally occurring beta-glucoside by a broad-specificity beta-glucosidase from liver.

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.