A novel nonhemorragic protease from the African puff adder (Bitis arietans) venom.

A nonhemorrhagic proteinase B-20 from the venom of Bitis arietans has been purified to apparent electrophoretic homogeneity by chromatography on Sephadex G-100, Q-Sepharose, and CM-cellulose. It has a molecular weight of 20 k Da as determined by size exclusion chromatography on Sephadex G-100 and migrated as a single 20-k Da band on SDS polyacrylamide. It has an optimum pH of 6-8 and is inactive at pH 4.0. EDTA and 1,10-phenanthroline strongly inhibited the enzyme suggesting it is a metalloenzyme. Also it is inhibited by antipain but is unaffected by trasylol, antitrypsin, and pepsptatin. Colombin, an identified active component of Aristolochia albida used in the treatment of snake poisoning, did not inhibit the protease activity. It lost over 90% of its activity in the presence of 0.5 microM Hg(2+) but the inhibition was completely blocked in the presence of 10 microM mercaptoethanol implicating sulfhydryl groups in the catalytic entity of the protein. The activity was also inhibited competitively by glutathione and cysteine with inhibition binding constants K(i) of 240 and 40 microM, respectively. The enzyme is unaffected by several divalent cations but activated by 1 mM Fe(3+). It had a prolyl endopeptidase and thermolysin-like activity. The enzyme displayed a fast acting alpha-fibrinolytic and delayed gamma-fibrinolytic activity when tested on human fibrinogen. The relevance of these findings is discussed.     

A cystatin-like cysteine proteinase inhibitor from venom of the African puff adder (Bitis arietans).

Venoms from eight snakes have been screened for inhibitory activity against papain, strong activity being found in that of the African puff adder, Bitis arietans. The inhibitor from B. arietans venom has been purified by affinity chromatography on carboxymethyl-papain-Sepharose and ion-exchange chromatography. The inhibitor had an apparent Mr of 13,000 in SDS/polyacrylamide gel electrophoresis, and pI value of 6.5 (major component) or 6.3 (minor component). Values of Ki for the inhibition of papain, cathepsin B and dipeptidyl peptidase I were 0.10, 2.7 and 0.23 nM, respectively; chicken calpain was not inhibited.     

Purification and properties of arginine esterases from Bitis arietans (puff adder) venom

An arginine esterase (FT1) was purified from B. arietans venom by gel-filtration and ion-exchange chromatography. The purified enzyme contains 21.6% of carbohydrate, 240 amino acids including 12 half-cystine residues and has a mol. wt of approximately 43,000. The purified enzyme has a high esterolytic activity towards N-alpha-benzoyl-L-arginine ethyl ester but shows no proteolytic activity against Azocoll and no clotting activity with fibrinogen. The N-terminal sequence of the arginine esterase from B. arietans venom shares a significant degree of sequence homology with the arginine esterase of B. nasicornis, the thrombin-like enzyme of C. adamanteus and the kallikrein-like enzymes of C. atrox venoms. It would appear that the arginine esterase from B. arietans venom exists in various multiple forms of the enzyme.     

A highly N-glycosylated chitin deacetylase derived from a novel strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 μmol min(-1) respectively.     

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.     

Aryl sulfatase from Naja nigricolis venom: characterization and possible contribution in the pathology of snake poisoning

The venom of Naja nigricolis was found to contain a high level of the enzyme aryl sulfatase. The enzyme was isolated from the venom of N. nigriclois and purified to electrophoretic homogeneity by gel chromatography on Sephadex G-100, DEAE-cellulose, and phenyl-sepharose columns. The enzyme was optimally active at pH 5 and 40 degrees C. Arrhenius plot for the determination of the activation energy (E(a)) gave the value 25 kJ/mol with a half-life (t(1/2)) of 5 min at 50 degrees C. It was highly activated by Fe(2+) and Ca(2+) and inhibited by Co(2+) and Mn(2+). The enzyme catalyzed the hydrolysis of the fluorescent compound methylumbelliferyl-sulfate (MU-SO(4)). Double reciprocal plots of initial velocity data, using MU-SO(4) as substrate, gave a K(M) value of 110 microM and V(max) of 225 micromol min(-1) x mg(-1). N. nigricolis Aryl sulphatase also hydrolyzed chondroitin-4-sulphate. It was inhibited competitively by N-acetyl glucosamine sulfate (GlcNAc-SO(4)), glucose-6-sulfate (Glc-6-SO(4)), and glucose 1-sulfate (Glc-1-SO(4)). Extrapolated inhibition binding constants (K(i)) gave the values of 3, 25, and 315 microM for GlcNAc-SO(4), Glc-6-SO(4), and Glc-1-SO(4) respectively.     

Purification and Properties of Dextransucrase from Streptococcus mutans

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.     

Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Purification and Characterization of Maleate Hydratase from Pseudomonas pseudoalcaligenes

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has been purified. The purified enzyme (98% pure) catalyzes the stereospecific addition of water to maleate and citraconate (2-methylmaleate), forming d-(+)-malate and d-(+)-citramalate, respectively. 2,3-Dimethylmaleate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxylic acids. The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. Maximum enzyme activity was measured at pH 8 and 45 degrees C. The K(m) for maleate was 0.35 mM, and that for citraconate was 0.20 mM. Thiol reagents, such as p-chloromercuribenzoate and iodoacetamide, and sodium dodecyl sulfate completely inhibited malease activity. Malease activity was competitively inhibited by d-malate (K(i) = 0.63 mM) and d-citramalate (K(i) = 0.083 mM) and by the substrate analog 2,2-dimethylsuccinate (K(i) = 0.025 mM). The apparent equilibrium constants for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2,050, 104, and 11.2, respectively.     

Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme

An olive (Olea europaea L.) β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified β-glucosidase was effectively active on p-/o-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) with K(m) values of 2.22 and 14.11 mM and V(max) values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by δ-gluconolactone and glucose against p-NPG as substrate. The K(i) and IC(50) values of δ-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with K(i) and IC(50) values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified β-glucosidase was investigated. Of the ions tested, only the Fe(2+) increased the activity while Cd(2+) Pb(2+) Cu(2+), Ni(+), and Ag(+) exhibited different levels of inhibitory effects with K(i) and IC(50) values of 4.29×10(-4) and 0.38×10(-4), 1.26×10(-2) and 5.3×10(-3), 2.26×10(-4) and 6.1×10(-4), 1.04×10(-4) and 0.63×10(-4), 3.21×10(-3) and 3.34×10(-3) mM, respectively.     

Synthesis of two diastereoisomeric p-nitrophenyl phosphodiesters of 2'',3''-secouridine and their affinity for phosphodiesterases.

The synthesis of the p-nitrophenyl esters of the 5'- and 3'-phosphates of the nucleoside analogue 2',3'-secouridine are described. Unlike the corresponding diesters of thymidine, these two compounds are diastereoisomers. Their affinity for phosphodiesterases types I and II were investigated. Both analogues were hydrolysed very slowly by snake venom phosphodiesterase but their affinity for the enzyme was similar to that of the p-nitrophenyl ester of thymidine 5'-monophosphate of which they were both competitive inhibitors with Ki approximately Km. Neither compound was hydrolysed by spleen phosphodiesterase but both competitively inhibited the p-nitrophenyl ester of thymidine 3'-monophosphate, with Ki's slightly higher than the Km. Although for each enzyme the Ki of the correct analogue phosphodiester (i.e. the 5'-derivative for snake venom and the 3'-derivative for spleen) was the lower, the absolute specificity seen for the normal substrates had been lost.     

Metabolism of 2-ketoaldehydes in mold: purification and characterization of glyoxalase I from Aspergillus niger

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.     

Effects of Some Metals on Carbonic Anhydrase from Brains of Rainbow Trout

Carbonic anhydrase (CA) enzyme was purified from rainbow trout brain by Sepharose-4B-L: -tyrosine-sulfanilamide affinity chromatography. The enzyme was obtained with a specific activity of 2,275 EU mg(-1) and a yield of 22.5%. The sample obtained from the affinity column was used for kinetic properties and inhibition studies. Both optimum and stable pH were found as 9.0 in 1 M Tris-SO(4) at 4 degrees C, respectively. To check the purity and subunit molecular weight of enzyme, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed, and MW was found as approximately 29.0 kDa. The molecular weight of native enzyme was estimated to be approximately 27.3 kDa by gel filtration chromatography. The purified enzyme had apparent K (m),V (max), and k (cat) as follows: 0.92 mM, 0.207 micromol.min(-1) and 43.6 s(-1) for p-nitrophenylacetate. The inhibitory effects of Co(II), Cu(II), Zn(II), Ag(I), and Cd(II) on CA enzyme activity were determined using the esterase method under in vitro conditions at low concentrations of the corresponding metals. The obtained IC(50) values, which cause 50% inhibition on in vitro enzyme activity, were 0.05, 30, 0.31, 159, and 82.5 mM for cobalt, copper, zinc, silver, and cadmium, respectively. K ( i ) values were also calculated from Linewaever-Burk plots for these substances as 0.014, 27.68, 2.15, 193.86, and 94.18 for cobalt, copper, zinc, silver, and cadmium, respectively; it was determined that cobalt, silver and cadmium inhibited the enzyme competitively, copper inhibited noncompetitively while zinc inhibited the enzyme uncompetitively.     

Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.     

Purification and characterizaton of plastidic pyruvate kinase from developing seeds of Brassica campestris L.

Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and non-competitively with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol.

The human pathogenic bacterium Clostridium difficile is a versatile organism concerning its ability to ferment amino acids. The formation of p-cresol as the main fermentation product of tyrosine by C. difficile is unique among clostridial species. The enzyme responsible for p-cresol formation is p-hydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficile strain DMSZ 1296(T) and initially characterized. The N-terminal amino-acid sequence was 100% identical to an open reading frame in the unfinished genome of C. difficile strain 630. The ORF encoded a protein of the same size as the purified decarboxylase and was very similar to pyruvate formate-lyase-like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzyme decarboxylated p-hydroxyphenylacetate (K(m) = 2.8 mM) and 3,4-dihydroxyphenylacetate (K(m) = 0.5 mM). It was competitively inhibited by the substrate analogues p-hydroxyphenylacetylamide and p-hydroxymandelate with K(i) values of 0.7 mM and 0.48 mM, respectively. The protein was readily and irreversibly inactivated by molecular oxygen. Although the purified enzyme was active in the presence of sodium sulfide, there are some indications for an as yet unidentified low molecular mass cofactor that is required for catalytic activity in vivo. Based on the identification of p-hydroxyphenylacetate decarboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermediates is proposed.     

Purification to homogeneity and properties of mannosidase II from mung bean seedlings

Mannosidase II was purified from mung bean seedlings to apparent homogeneity by using a combination of techniques including DEAE-cellulose and hydroxyapatite chromatography, gel filtration, lectin affinity chromatography, and preparative gel electrophoresis. The release of radioactive mannose from GlcNAc[3H]Man5GlcNAc was linear with time and protein concentration with the purified protein, did not show any metal ion requirement, and had a pH optimum of 6.0. The purified enzyme showed a single band on SDS gels that migrated with the Mr 125K standard. The enzyme was very active on GlcNAcMan5GlcNAc but had no activity toward Man5GlcNAc, Man9GlcNAc, Glc3Man9GlcNAc, or other high-mannose oligosaccharides. It did show slight activity toward Man3GlcNAc. The first product of the reaction of enzyme with GlcNAcMan5GlcNAc, i.e., GlcNAcMan4GlcNAc, was isolated by gel filtration and subjected to digestion with endoglucosaminidase H to determine which mannose residue had been removed. This GlcNAcMan4GlcNAc was about 60% susceptible to Endo H indicating that the mannosidase II preferred to remove the alpha 1,6-linked mannose first, but 40% of the time removed the alpha 1,3-linked mannose first. The final product of the reaction, GlcNAcMan3GlcNAc, was characterized by gel filtration and various enzymatic digestions. Mannosidase II was very strongly inhibited by swainsonine and less strongly by 1,4-dideoxy-1,4-imino-D-mannitol. It was not inhibited by deoxymannojirimycin.