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1.
Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase. 相似文献
2.
Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides 总被引:7,自引:4,他引:3
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Gunilla Bengtsson Thomas Olivecrona Magnus Hk Johan Riesenfeld Ulf Lindahl 《The Biochemical journal》1980,189(3):625-633
1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40–50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin–Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro. 相似文献
3.
Yoram Salomon Yehudith Amir Riki Azulai Abraham Amsterdam 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(2):262-272
Heparin inhibits (I50 = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I50 = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation. 相似文献
4.
Paramita Chaudhuri Arghya BasuShinjinee Sengupta Sagar LahiriTrina Dutta Anil K. Ghosh 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high Vmax and a low Km value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl2 and ZnCl2 acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH. 相似文献
5.
Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15 总被引:1,自引:0,他引:1
Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I. 相似文献
6.
Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium 总被引:2,自引:0,他引:2
A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources. 相似文献
7.
L. C. Chen C. Y. Wu C. F. Chen C. F. Chiang 《Preparative biochemistry & biotechnology》2013,43(4):183-195
Abstract The chlorpromazine-sensitive GTPase from the cell membrane of rat cerebral cortex was purified to homogenity by using DEAE Bio-Gel A agarose, hydroxyapatite and heparin agarose chromatography. The purified chlorpromazine-sensitive GTPase was purified 370-fold to obtain a final specific activity of 40 nmol GTP hydrolyzed/min/mg protein. The purified enzyme was inhibited by chlorpromazine but not by compound 48/80. Magnesium was required for its activity instead of calcium. The purified enzyme had an apparent pH optimum of 8.0, and molecular weight was estimated to be 58,000. 相似文献
8.
The effect of the polyanion heparin on the trehalose phosphate synthetase of Mycobacterium smegmatis had been studied. In the presence of heparin (0.5 mg/ml), the synthetase shows greatly increased stability when heated at 50 °C for various periods of time as compared to the enzyme in the absence of heparin. Heparin also prevents digestion of the enzyme by trypsin. In the absence of heparin, the synthetase is retained on a Sephadex G-200 column and elutes in an area suggesting a molecular weight of about 40,000–50,000. However, when heparin (0.5 mg/ml) is mixed with the enzyme, the synthetase is excluded from the Sephadex G-200 column and elutes in an area suggesting a molecular weight of greater than 450,000. The trehalose phosphate synthetase was purified by binding it to a column of heparin covalently attached to Sepharose 4B. The synthetase was eluted from this column with a linear gradient of heparin. This enzyme fraction which contained bound heparin showed greatly increased stability at 50 °C, and eluted from the Sephadex G-200 column in an area suggesting a molecular weight of greater than 450,000. These results indicate that heparin, and presumably other polyanions, stabilizes the synthetase to adverse conditions and also causes an association of the enzyme to high molecular weight forms.The synthetase, when bound to the heparin-Sepharose gel, still retained good enzymatic activity. This immobilized enzyme was active with various glucose sugar nucleotides (ADP-glucose, GDP-glucose, UDP-glucose, TDP-glucose) and did not require additional polyanion. The product formed from each of these sugar nucleotides was shown to be trehalose phosphate by a variety of chemical and enzymatic procedures. 相似文献
9.
Heparinase I from Flavobacterium heparinum, a source of diverse polysaccharidases, suffers from low yields, insufficient purity for structural studies and insolubility when expressed as a recombinant product in Escherichia coli that is devoid of glycosaminoglycan polysaccharidases. In this study, cDNA coding for the orthologue of F. heparinum heparinase I was constructed from genomic information from the mammalian gut symbiont Bacteroides thetaiotaomicron and expressed in E. coli as a fusion protein with GST at the N-terminus. This resulted in high yield (30 mg/g dry bacteria) of soluble product and facilitated one-step affinity purification to homogeneity. Purified heparinase I bearing the GST fusion exhibited a Km of 2.3 μM and Vmax of 42.7 μmol/min with a specific activity of 164 U/mg with heparin (average 12,000 Da) as substrate. The results indicate a 2-fold improvement in yield, specific activity and affinity for heparin as substrate over previous reports. The data suggest that the heparinase I from the gut symbiont exhibits a higher intrinsic affinity for heparin than that from F. heparinum. The purified GST fusion enzyme exhibited a requirement for Ca2+ and a pH optimum between 6.7 and 7.3 that was similar to the enzyme freed of the N-terminal GST portion. Our study revealed that catalytic activity of heparinase I requires a reducing environment. The GST facilitated immobilization of heparinase I in solid phase either for clinical purposes or for structural studies in absence of interference by contaminating polysaccharidases. 相似文献
10.
Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15. 总被引:1,自引:0,他引:1
Sung-Woon Hong Ho-Young Shin Yeong Shik Kim Dong-Hyun Kim 《European journal of biochemistry》2003,270(15):3168-3173
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. 相似文献
11.
Paul J. Geutjes Suzan T.M. Nillesen Gerwen Lammers Willeke F. Daamen Toin H. van Kuppevelt 《Protein expression and purification》2010,69(1):76-82
Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation (~124 mg/L) and purified by heparin affinity chromatography. SDS–PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48 kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3 days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences. 相似文献
12.
A cytolytic protein (perforin) was rapidly purified from a cell line of mouse cytotoxic T-lymphocytes (CTL) by DEAE-cellulose, heparin-Sepharose, and phenyl-Sepharose chromatographies. The purified perforin was activated by heparin, the half maximal concentration being 3-10 ng/ml, depending on the calcium concentration. Other acid mucopolysaccharides, such as chondroitin sulfates A and C, keratan polysulfate, and heparin sulfate, also enhanced the lysis of erythrocytes by perforin, but the concentrations required for activation were more than 100-fold higher than that of heparin. Chondroitin, hyaluronic acid, and keratan sulfate, however, had no effect on the perforin activity. It was suggested that heparin potentiates the lytic activity of perforin and acid mucopolysaccharides may actually be involved in target cell lysis by CTL. 相似文献
13.
Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions 总被引:1,自引:0,他引:1
The trehalose-P synthase was purified to near homogeneity fromthe cytoplasmic fraction of Mycobacterium smegmatis. At thefinal stage of purification, the enzyme preparation showed onemajor band of 59 kDa on SDS gels. The 59 kDa band became labeledwith N3-UDP[32P]-glucose, and this labeling was inhibited ina concentration-dependent manner by either unlabeled UDP-glucoseor GDP-glucose. The native enzyme also had a molecular weightof about 60 kDa by gel filtration, indicating that the activeenzyme is a monomer. The 59 kDa protein was subjected to endoproteinaseLys-C digestion, and three peptides isolated by HPLC were sequenced.The sequences of 56 amino acids in these three peptides showed60% identity to the trehalose-P synthases of Saccharomyces cerevesiaeand Schizosaccharomyces pombe. The purified mycobacterial enzymecatalyzed the synthesis of trehalose-P from glucose-6-P anda variety of nucleoside diphosphate glucose derivatives, dependingon whether a polyanion was absent or present. Thus, UDP-glucoseand GDP-glucose were the best glucosyl donors, but maximum activitywith UDP-glucose required the presence of a polyanion such asheparin, whereas activity with GDP-glucose was relatively independentof polyanion. The presence of heparin in the incubation mixtureincreased the affinity of the enzyme for UDP-glucose by a factorof 100, or more. However, the affinity for GDP-glucose was onlytwofold better in the presence of heparin. The purified synthasealso utilized ADP-glucose and CDP-glucose, but the Km for theseglucosyl donors was quite high even in the presence of polyanion.The effect of heparin on UDP-glucose activity was dose-dependentand maximum at about 12 µ;g of heparin/incubation.However, the size of the heparin molecule (i.e., the numberof monosaccharide residues) was critical for activation, andonly those heparins with 18 or more monosaccharide units wereeffective in stimulating activity. trehalose polyanions mycobacteria GDP-glucose heparin 相似文献
14.
The interaction of sulfated mucopolysaccharides and lectins has been studied by determining the amount of precipitate formed when mucopolysaccharides are added to a solution of concanavalin A or a partially purified lectin preparation from red kidney bean (Phaseolus vulgaris). The amount of insoluble complex obtained when a given mucopolysaccharide is added to a solution of partially purified red kidney bean preparation is pH dependent. The reaction of concanavalin A and heparin has also been studied by adding increasing amounts of mucopolysaccharide to a fixed amount of lectin. This interaction results in the development of a precipitin-like curve and leads to the isolation of a heparin fraction which has been found to be more reactive with respect to formation of a precipitate than the original heparin preparation. Monosaccharides such as α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine which are known to bind specifically to the lectin, greatly inhibit precipitate formation. The interactions between sulfated mucopolysaccharides and lectins have been used to isolate various sulfated mucopolysaccharides. 相似文献
15.
Purification and properties of an endothelial cell growth factor from human platelets 总被引:26,自引:0,他引:26
K Miyazono T Okabe A Urabe F Takaku C H Heldin 《The Journal of biological chemistry》1987,262(9):4098-4103
An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin. 相似文献
16.
采用HNO2降解法,通过控制降解过程中的pH和时间裂解低分子肝素,并经大孔离子交换树脂BS-Ⅱ对其进行分离纯化,用质谱法对其相对分子质量进行分析。结果表明:在pH3.5、4 h的降解条件下,降解得到低分子肝素。在0.34 mol/L NaCl、pH8.5的条件下吸附,4 mol/L NaCl、pH8.5条件下洗脱,其吸附率达97.2%,纯化后的低分子肝素抗凝活性为34.41 U/mg,比纯化前提高1.36倍,效价回收率达到85.24%。质谱分析表明,纯化所得低分子肝素的平均相对分子质量为8 411,67.45%集中分布在7 000~9 000段。 相似文献
17.
Kinzo Nagasawa Hideki Uchiyama 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(2):430-440
Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl 0.12 mol; anticoagulant activity, 117 USP units/ mg) was reacted with 5-isothiocyanatofluorescein in 0.5M carbonate buffer (pH 8.5) at 35°C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (λem 516 nm, λex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively).The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting the assay were virtually identical. 相似文献
18.
To determine N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) in human neutrophil granules separated by a method requiring heparin, the inhibition of this enzyme by heparin was studied. Neutrophils were purified from blood of five donors by modifications of the Hypaque-Ficoll and dextran separation methods resulting in a suspension which was 96% neutrophils. Neutrophil lysates were assayed for N-acetyl-beta-D-glucosaminidase by measuring the amount of p-nitrophenol released from p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The reaction showed first-order kinetics with regard to enzyme concentration. Triton X-100, 0.1% v/v, enhanced enzyme activity. Heparin was shown to reduce neutrophil lysate N-acetyl-beta-D-glucosaminidase to a specific activity of 46% at a heparin concentration of 2 units per assay and to 43% (maximal inhibition) at 17 and 50 units of heparin per assay. Substantially higher heparin concentrations partially restored the inhibited activity, the maximal restoration being a return to 80% of the original activity at 1700 units of heparin per assay. Protamine sulfate was assessed for its ability to restore N-acetyl-beta-D-glucosaminidase activity in the presence of heparin. At 1.0 mg/10 units of heparin, protamine restores enzyme activity to its heparin-free activity. These studies of human neutrophil N-acetyl-beta-D-glucosaminidase demonstrate: (1) specific enzyme activity is 28.8 +/- 7.0 nmole p-nitrophenol released per minute per milligram of protein or 1.7 +/- 0.5 nmole p-nitrophenol released per minute per 10(6) neutrophils; (2) heparin rapidly but finitely inhibits enzyme activity at very low concentrations and paradoxically restores it toward normal at high concentrations; and (3) protamine sulfate restores enzyme activity inhibited by heparin. 相似文献
19.
《Phytochemistry》1986,25(11):2489-2492
An acid endoribonuclease isolated from corn leaf tissues was purified 530 times. Gel electrophoresis indicated that the enzyme was homogeneous. The enzyme showed an optimum pH at 5.5 and an apparent molecular weight of 32 000. Corn RNase attacks natural RNAs and synthetic polyribonucleotides and the relative rate of degradation was poly U > yeast RNA > E. coli tRNA > poly A ⪢ poly C. Zn2+, Mg2+, Mn2+ and EDTA inhibited the enzyme activity. No stimulation by K+ was observed. Cu2+ and heparin had no effect on the activity. The results suggest that the investigated RNase differs from other known corn ribonucleases. 相似文献
20.
Fucosylated chondroitin sulfate (FCScs) isolated from sea cucumber Cucumaria syracusana was characterized by Fourier Transform InfraRed spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy and high performance size exclusion chromatograph, a multi-angle laser light scattering detector, a viscometer and a differential refractive index (dRI) detector (HPSEC-MALLS-dRI). The anticoagulant activities of FCScs were studied by the classical clotting time assays and the purified systems containing thrombin and antithrombin or heparin cofactor II. The effect on thrombin generation was investigated using calibrated automated thrombography (CAT). The results obtained showed that the FCS with high sulfate content 31 % and relatively low average molecular weight of 36.3 kDa was isolated from C. syracusana in amount of ∼ 35.6 mg/g dry body wall. Structural analysis of this polysaccharide revealed the presence backbone structure of chondroitin sulfate chain branched by two types of fucose 2,4-O-di and 3,4-O-disulfated residues in respective ratios of 57.5 and 42.5 %. The FCScs exhibited a high anticoagulant activity mediated essentially by heparin cofactor II (HCII) and to lesser extent by antithrombin (AT) with IC50 values of 0.05 μg/mL and 0.09 μg/mL, respectively. Furthermore, the results of CAT assay showed that the velocity index decreases 3-times at 50 μg/mL in comparison with normal plasma. The overall results showed high anticoagulant activity attributed to the high sulfate content and abundance of disulfated fucose branches of FCScs which made it a promising candidate of anticoagulation drug. 相似文献