Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin.

1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. The KD for high molecular weight HA was 5.4 10(-9) M at 37 degrees C and lower than 10(-10) M at 4 degrees C. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. The specific site for interaction was the HA-derived decasaccharide HA10. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage.     

Caenorhabditis elegans: purification of isocitrate lyase and the isolation and cell-free translation of poly(A+)RNA

Isocitrate lyase (EC 4.1.3.1) from mixed larval populations of Caenorhabditis elegans was stabilized in crude extracts by centrifugation over a 0.2-0.6 M sucrose gradient for 2.5 hr in a vertical rotor (VTi 50) at 210,000g. The peak fractions from this sucrose gradient showed a half-life of 33 hr at 30 C and 225 hr at 4 C in contrast to 2.5 and 52 hr, respectively, for the crude extract. A purification scheme involving (NH4)2SO4 precipitation and chromatography on Sepharose 6B and diethylaminoethyl-cellulose yielded isocitrate lyase that gave one band after electrophoresis in a sodium dodecyl sulfate-gel polymerized from 12% acrylamide. The purified enzyme with a molecular weight of 250,000 and subunit molecular weight of 61,600, had a specific activity of 2 mumoles glyoxylate formed min-1 mg protein-1, and was obtained in a 4% yield. Isocitrate lyase from C. elegans lost 80-85% of its activity in the precipitation by 33-55% (NH4)2SO4, but this step appeared to be necessary for purification to homogeneity. The use of fast protein liquid chromatography appeared to be promising in that it provided an enzyme preparation that was about 50% pure with a specific activity as high as 3 mumoles glyoxylate formed min-1 mg protein-1. Poly(A+)RNA was isolated from C. elegans and translated in wheat germ cell-free system. Analysis on a 10% sodium dodecyl sulfate-polyacrylamide gel showed varied translation products including one or more 60,000-Da polypeptides.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Effect of oxygen and shear stress on molecular weight of hyaluronic acid

Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production, yet various views persist about their effect on the molecular weight of HA. Accordingly, this study investigated the effects of DO and shear stress during HA fermentation. The results showed that both cell growth and HA synthesis were suppressed under anaerobic conditions, and the HA molecular mass was only (1.22+/-0.02) x 106 Da. Under aerobic conditions, although the DO level produced no change in the biomass or HA yield, a high DO level favored the HA molecular mass, which reached a maximum value of (2.19+/- 0.05) x 106 Da at 50% DO. Furthermore, a high shear stress delayed the rate of HA synthesis and decreased the HA molecular weight, yet had no clear effect on the HA yield. Therefore, a high DO concentration and mild shear environment would appear to be essential to enhance the HA molecular weight.     

The signal-to-noise ratio as a measure of HA oligomer concentration: a MALDI-TOF MS study

MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine ng amounts of defined hyaluronan (HA) oligomers obtained by enzymatic digestion of high molecular weight HA with testicular hyaluronate lyase. The signal-to-noise (S/N) ratio of the positive and negative ion spectra represents a reliable concentration measure: Amounts of HA down to about 40 fmol could be determined and there is a linear correlation between the S/N ratio and the HA amount between about 0.8 pmol and 40 fmol. However, the detection limits depend considerably on the size of the HA oligomer with larger oligomers being less sensitively detectable. The advantages and drawbacks of the S/N ratio as concentration measure are discussed.     

Two forms of alpha-amylase in mantle tissue of Mytilus galloprovincialis: purification and molecular properties of form II

alpha-Amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. alpha-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis alpha-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 degrees C.     

Properties of Alginate Lyases from Marine Bacteria

Alginate lyases (EC 4.2.2.3) from two marine bacteria were isolated and partially characterized. A cell-bound lyase from isolate A3 had a molecular weight of approximately 100,000 and cleaved mannuronate blocks, apparently in an exo manner. A lyase recovered from the culture medium of isolate W3 was soluble in saturated ammonium sulfate, cleaved guluronate blocks, apparently in an endo manner, and had a molecular weight of 35,000. The thiobarbiturate test and urea-polyacrylamide gel electrophoresis were used to determine substrate specificity and mode of substrate cleavage by the enzymes.     

Immunolocalization of cholesterol side chain cleavage enzyme (P450scc) in <Emphasis Type="Italic">Mytilus </Emphasis><Emphasis Type="Italic">galloprovincialis</Emphasis> and its induction by nutritional levels

We have examined the localization of P450scc in different tissues of the mollusc Mytilus galloprovincialis along a gonadal cycle by using a polyclonal antibody against rat cholesterol side-chain cleavage enzyme (cytochrome P450scc). Immunoreactivity specific for P450scc was found only in the cytoplasm of basophilic cells from digestive gland; gonad, gills and kidney appeared to be devoid of P450scc immunoreactivity. SDS-gel electrophoresis and western blot analysis of different subcellular fractions revealed that this protein is mainly located in microsomes, has a molecular weight of 51.4 kDa and is recognized by the antibody against rat P450scc. These results suggest that the digestive gland of M. galloprovincialis could be considered a steroidogenic tissue where the first step in biosynthesis of steroid hormones occurs. Moreover, seasonal slot blot analysis of post-mitochondrial fractions showed a P450scc induction by available nutrients and phytoplankton blooms.     

Purification and characterization of an extracellular pectate lyase from an Amycolata sp.

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.     

Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15.

Salt-active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with GAG degrading enzymes. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, diethylaminoethyl (DEAE)-cellulose, CM-Sephadex C-50, HA ultrogel and phosphocellulose column chromatography with the final specific activity of 81.33 micro mol x min-1 x mg-1. The purified salt-active acharan sulfate lyase was activated to 5.3-fold by salts (KCl and NaCl). The molecular weight of salt-active acharan sulfate lyase was 94 kDa by SDS/PAGE and gel filtration. The salt-active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 degrees C. Salt-active acharan sulfate lyase activity was potently inhibited by Cu2+, Ni2+ and Zn2+. This enzyme was inhibited by some agents, butanediol and p-chloromercuric sulfonic acid, which modify arginine and cysteine residues. The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate, to a lesser extent on heparan sulfate and heparin. The biochemical properties of the purified salt-active acharan sulfate lyase are different from those of the previously purified heparin lyases. However, these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II.     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

Hyaluronic acid promotes chick embryo fibroblast and chondroblast expression

15-day-chick-embryo fibroblasts and chondroblasts were cultured in the presence of high and low molecular weight exogenous hyaluronic acid (HA). Growth range and incorporation of radiolabelled sulphate and proline were determined. HA reduced cell proliferation to about 75% of controls, while incorporation of radiolabelled sulphate and proline was higher in HA-treated cultures of both chondroblasts and fibroblasts. The effect was not due to the polyanionic or polymeric nature of the molecule and appeared to be highly specific for HA.     

Purification of a factor inhibiting differentiation from conditioned medium of nondifferentiating mouse myeloid leukemia cells

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-factor activity) was detected in conditioned medium of variant M1 cell clones that were resistant to differentiation inducers, and this I-factor activity was shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-factor was purified to apparent homogeneity from conditioned medium of resistant M1 cells. The purification procedure consisted of ammonium sulfate precipitation, CM-Sepharose CL-6B, Sephadex G-200, reverse-phase high performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The factor was analyzed by radioiodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The purified factor gave a single band of protein with a molecular weight of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with its biological activity. The concentration of I-factor required for 50% inhibition of dexamethasone-induced differentiation of M1 cells was 24 pM. At its effective concentration it had no effect on cell proliferation, and even at 1.2 nM it did not inhibit colony formation of normal bone marrow cells, suggesting that it was distinct from the inhibitor of normal precursors of macrophages and/or granulocytes.     

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.     

Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100 °C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.     

The Estimation of the Quantity of Isocitrate Lyase Protein in Acetate-Adapted Cells of Chlorella pyrenoidosa

The quantity of isocitrate lyase protein was estimated, as apercentage of cell dry weight, by three different electrophoreticmethods: (a) direct collection and determination of proteinsafter electrophoresis; (b) separation and estimation of ₃₅S-labelledproteins; (c) estimation from the density of stained bands onacrylamide gels. The possibility that protein-protein interactionduring electrophoresis might interfere with the results wasconsidered and discounted. The average result from the threemethods is that, in acetate-adapted cells of Chlorella pyrenoidosa,isocitrate lyase protein constitutes about 7.0 per cent of totalsoluble proteins (100,000 g supernatant), that is 1.0 per centof cell dry weight. The estimate agrees well with one basedon the increase in specific activity of the enzyme during purification.     

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum.

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.     

Self-association of hyaluronate segments in aqueous NaCl solution

The potential for self-association by hyaluronate (HA) chains in 0.15 M NaCl was investigated, using low molecular weight HA segments as a model system. HA segments were derived from the polymer by controlled enzymatic digestion, and purified by gel filtration chromatography. Seven samples of narrow molecular weight distribution were analyzed by sensitivity-enhanced polyacrylamide gel electrophoresis, and found to have the following weight-average numbers of repeating disaccharide units: A, 90; B, 51; C, 38; D, 31; E, 23; F, 18; G, 13. The segment preparations were studied in 0.15 M NaCl by capillary viscometry, low angle laser light scattering, and circular dichroism spectroscopy. The data indicate concentration-dependent intermolecular association of short segments, and a capability for intramolecular association (hairpin formation) by larger HA segments.     

The cell wall of the pathogenic bacterium Rhodococcus equi contains two channel-forming proteins with different properties

We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.     

Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.