Isolation and characterization of a sialidase from the starfish Asterias rubens

The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.     

Properties of sialidase isolated from Actinomyces viscosus DSM 43798

The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.     

Human placental sialidase: partial purification and characterization

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.     

Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257

A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Purification and kinetic properties of sialidase from Clostridium perfringens

Clostridium perfringens sialidase was isolated from a culture medium of bacterial cells by ammonium sulfate precipitation (42-85%), followed by purification through Sephadex G-75 gel chromatography, DEAE A-50 anion exchange chromatography, FPLC medium pressure anion exchange chromatography and finally FPLC medium pressure isochromatofocussing. From 9 l culture medium 1.17 mg sialidase was isolated with a specific activity of 295 U/mg. The enzyme appeared to be homogeneous by analytical polyacrylamide gel electrophoresis. The molecular mass was measured to be 66 kDa. Km values ranging from 0.6 to 1.6mM were determined for several oligosaccharides as substrates. The enzyme catalyzed transglycosylation reactions with methanol as a nucleophilic reagent competitive with water. In the enzymatic hydrolysis of the (3'-methoxyphenyl)glycoside of alpha-N-acetylneuraminic acid, increase of methanol concentration had no effect on the release of 3-methoxyphenol. This finding suggests that the formation of the enzyme-glycon intermediate is the rate-determining step for this substrate.     

Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase.

Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity was solubilized by the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulphonate (CHAPS), whose presence was necessary for maximal activity. Purification included Q-Sepharose ion-exchange chromatography, preparative isoelectric focusing and HPLC gel filtration on TSK 3000 matrix. Overall purification from post-nuclear supernatants was estimated to be 110,000-fold with a 50% recovery of activity. The purified enzyme hydrolysed Man9GlcNAc1,2 from thyroglobulin or oligosaccharide-lipid, but not invertase Man9GlcNAc, Man1 alpha 2Man1 alpha OCH3 or p-nitrophenyl-alpha-D-mannopyranoside. Conversion of thyroglobulin Man9GlcNAc to Man8GlcNAc was linear with time and enzyme concentration, with an apparent Km of 0.2 mM and a specific activity of 220 IU/mg. Glc3Man9GlcNAc2 from oligosaccharide-lipid was as good a substrate as Man9GlcNAc, but the lipid-linked Man7GlcNAc2 isomer was hydrolysed at only 10% of this rate. Hydrolysis of defined isomers of IgM and bovine thyroglobulin Man6,7,8GlcNAc indicated that, for maximal alpha 1,2-mannosidase activity, only the alpha 1,2-linked terminal mannoses on the alpha 3 branch of the Man9GlcNAc precursor were dispensable. Isomers lacking the terminal alpha 1,2-linked mannose on the alpha 6 branch were hydrolysed at only approximately 10% of the maximal rate. The enzyme exhibited a pI of 5.3 and a pH optimum at 6.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents gave a single sharp band at 66 kDa, while in the presence of beta-mercaptoethanol equimolar amounts of two peptides, one of 44 kDa and one of 23 kDa, were obtained. Sizing on Sephacryl SF300, Superose 12 and TSK 3000 provided a holoenzyme mol. wt of 60-68 kDa, indicating that the isolated active form of the Man9-alpha-mannosidase was composed of one each of the sulphydryl-bonded dissimilar peptides. The enzyme bound to concanavalin A (ConA)-Sepharose and was eluted with alpha-methylmannoside, indicating the presence of high-mannose oligosaccharides. The Man9-alpha-mannosidase required low levels of Ca2+, which could be removed by EGTA. Activity was restored by Ca2+ or Zn2+, but not by Mg2+ or Mn2+.     

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.     

Purification and characterization of a cytosolic Ca<Superscript>2+</Superscript>-independent phospholipase A<Subscript>2</Subscript> from bovine brain

The Ca²⁺-independent phospholipase A₂ (iPLA₂) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA₂ from bovine brain. iPLA₂ was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA₂ activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on twodimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃), Triton X-100, iron, and Ca²⁺. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA₂, and adenosine triphosphate (ATP). The spot with the iPLA₂ activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA₂.     

Purification and N-terminal amino acid sequence of fructose-6-phosphate phosphoketolase from Bifidobacterium longum BB536

AIMS: The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 4.1.2.22.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536). METHODS AND RESULTS: A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 +/- 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 +/- 1 kDa and 59 +/- 0.5 kDa, as determined by SDS-PAGE. CONCLUSION: The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.     

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene.

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.     

Isolation and some properties of lysineN 6-hydroxylase fromEscherichia coli strain EN222

Summary Lysine N⁶-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication ofEscherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium sulfate precipitation and fast protein liquid chromatography using Superose 12 and Mono Q columns. The molecular mass as determined by gel filtration was 200 kDa and 50 kDa by SDS/polyacrylamide gel electrophoresis. The enzyme binds 0.79 molecule FAD/50 kDa. The activity of the enzyme is strictly dependent on NADPH. Its properties are similar to other flavoprotein monooxygenases of the EC group 1.14.13.     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.     

长双歧杆菌α-唾液酸苷酶在大肠杆菌中的表达及酶学性质

[背景]唾液酸苷酶是一类水解唾液酸糖复合物末端唾液酸残基的糖苷水解酶,广泛存在于动物和微生物中,具有重要的生物学功能.[目的]克隆一个长双歧杆菌(Bifidobacterium longum)唾液酸苷酶基因(blsia42)并在大肠杆菌(Escherichia coli)中表达,探讨该重组酶的酶学性质.[方法]从长双歧...     

A sialidase complex from chicken liver: Characterization of a multienzyme complex with β-galactosidase and carboxypeptidase

Mammalian lysosomal sialidase exists as an enzyme complex with β-galactosidase and carboxypeptidase, so-called “protective protein.” In this article, we report that chicken sialidase also occurs as a complex with β-galactosidase and protective protein. The purified sialidase complex had a molecular weight > 700 kDa on gel filtration and showed four protein components of 76, 65, 54 and 48 kDa on SDS-PAGE under nonreducing conditions. N-Terminal sequences of the 65- and 48-kDa proteins were homologous to human lysosomal β-galactosidase and protective protein precursor, respectively. The purified sialidase complex also had carboxypeptidase activity. Both sialidase and carboxypeptidase activities were precipitated together by an antibody against chicken β-galactosidase. The complex reversibly dissociated into 120-kDa β-galactosidase dimer and 100-kDa carboxypeptidase dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. These findings indicate that chicken sialidase exists as a multienzyme complex, by which the sialidase activity appears to be stabilized.     

2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Purification and some properties of the enzyme

A novel aerobic mechanism of 2-aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2-Aminobenzoic acid is activated in a coenzyme-A-ligase reaction to 2-aminobenzoyl-CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2-aminobenzoyl-CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown non-aromatic compound. 2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 mumol.min-1.mg-1 protein using ammonium sulfate precipitation, DEAE-cellulose anion-exchange, hydroxylapatite and Mono Q FPLC anion-exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an alpha 2 dimer; however, it may exist in three dimeric forms, alpha alpha, alpha alpha' and alpha' alpha', where alpha' may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2-aminobenzoyl-CoA (Km less than or equal to 25 microM) and O2 (Km less than or equal to 5 microM), but less specific for the reduced pyridine nucleotides NADH (Km = 42 microM) or NADPH [Km = 500 microM; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min-1. The enzyme also reduced N-ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.     

Isolation and characterization of a xylose-glucose isomerase from a new strain Streptomyces thermovulgaris 127, var. 7-86.

A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Human placental sialidase: further purification and characterization

An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.