Purification and characterization of acid phosphatase-1 from Drosophila melanogaster

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified to apparent homogeneity. The final product is a glycoprotein homodimer with a subunit molecular weight of about 50,000, as measured by its electrophoretic mobility in denaturing conditions on polyacrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37 degrees C, pH 5.0. An average fly contains about 5 ng of enzyme. Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3. The enzyme hydrolyzes a wide variety of phosphate monoesters, including AMP, glucose 6-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high substrate concentrations. The Michaelis constants for 1-naphthyl phosphate and p-nitrophenyl phosphate are 79 microM and 68 microM, respectively, at pH 5.0 and 37 degrees C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10 degrees C rise in temperature. Above 48 degrees C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Staphylococcal acid phosphatase: preliminary physical and chemical characterization of the loosely bound enzyme

At temperatures between 45 and 50 C, staphylococcal acid phosphatase purified 44-fold had maximal activity at pH 5.2 to 5.3. However, the enzyme was most stable in the alkaline range (pH 8.5 to 9.5) at temperatures below 50 C. Iodoacetate and ethylenediamine-tetraacetic acid were effective inhibitors, whereas mercaptoethanol and Cu(2+) acted as stimulators. The energy of activation for hydrolytic cleavage of the synthetic substrate, p-nitrophenyl phosphate, was 19.5 Kcal/mole. K(m) for the same substrate was 4.5 x 10(-4)m. The purified enzyme was most active against the substrates p-nitrophenyl phosphate and glyceraldehyde 3-phosphate.     

Purification and physicochemical characterization of a human placental acid phosphatase possessing phosphotyrosyl protein phosphatase activity

A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.     

13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.     

Prolipoprotein modification and processing enzymes in Escherichia coli

Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate. This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope. Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM. Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity. The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively. Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using [2-3H]glycerol-labeled JE5505 cell envelope and [35S]cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively. 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction. The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.     

Purification and characterization of phosphodiesterase from Crotalus venom.

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.     

The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1-3 mM Ca2+, and at temperatures near 37 degrees C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Characterization of Adenosine deaminase (ADA) in hemolymph of Biomphalaria glabrata.

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 micromols.L(-1). Highest activity was found at 37 degrees C. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 microL of pure hemolymph per assay, pH 6.8, and 37 degrees C. This enzyme showed activities of 834 +/- 67 micromol.min(-1).L(-1) (25 degrees C) and 2029 +/- 74 micromol.min(-1).L(-1) (37 degrees C), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43 degres C or 70% at 50 degrees C for 15 minutes. The ADA lost from 26% to 78% of its activity when hemolymph was pre-incubated at 50 degrees C for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.     

Purification and characterization of a chitinase from Trichoderma viride

Usukizyme, a commercial enzyme preparation from Trichoderma viride, showed multiple chitin- degrading activities. One of these was purified to homogeneity by sequential DEAE Sepharose CL-6B, Q-Sepharose FF, and Sephacryl S-100 HR column chromatographies. The purified enzyme showed optimum activity at pH 3.5 and 50 degrees -55 degrees C and was stable in the pH range of 3.5-6.0 and up to 45 degrees C. It showed higher activity toward chitosan-7B, a 62% deacetylated chitosan, as opposed to highly deacetylated chitosan substrates. Products of degradation of a 1% (w/v) solution of partially deacetylated chitin (PC-100) were purified on CM-Sephadex C-25 and analyzed by HPLC, exo-glycosidase digestion, and nitrous acid deamination. The enzyme was unable to split the GlcN-GlcN linkages in the substrate. It produced mainly (GlcNAc)(2) and (GlcNAc)(3) along with mixed oligosaccharides. When subjected to nitrous acid degradation, some of the mixed oligosaccharides produced mainly 2-deoxyglucitol, implying the presence of GlcN at the reducing end of the oligosaccharides.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Purification and characterization of a phytase from Pseudomonas syringae MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40 degrees C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.     

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min(-1) mg(-1) protein and had a Km(avg) of 235 microM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu(2+) and Mg(2+); (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37-80 degrees C, with maximum activity at 37 degrees C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI approximately 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.     

Characterization of turkey pancreatic lipase

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.     

Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6.3.1.2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smegmatis in the subunit size (56,000), molecular weight (670,000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45 degrees C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.     

Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

Bovine brain low Mr acid phosphatase: purification and properties

Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.     

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.