Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii

A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.     

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).     

Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.     

Purification and characterization of an exoglucanase from Streptomyces flavogriseus

Streptomyces flavogriseus, a mesophilic actinomycete, produces high levels of extracellular enzymes capable of hydrolyzing cellulose and xylan. One such enzyme, an exoglucanase, has been purified to molecular homogeneity by a sequence involving DEAE Bio-Gel A chromatography, gel permeation chromatography on Bio-Gel P-60, preparative isoelectric focusing, and concanavalin A affinity chromatography. This purification sequence disclosed the presence of several distinct endoglucanase and xylanase fractions. Homogeneity of the purified enzyme was demonstrated by analytical isoelectric focusing and sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 45 000 and an isoelectric point of 4.15. The enzyme demonstrated negligible activity with carboxymethylcellulose as the substrate. It was able to extensively hydrolyse acid-swollen cellulose; the main product of enzyme action was cellobiose.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Heat shock response in Neurospora crassa: purification and some properties of HSP 80

The heat shock response of Neurospora crassa was investigated. A 80-kilodalton heat shock protein (HSP 80) was purified to near homogeneity from heat-shocked mycelial extracts employing ammonium sulphate fractionation, gel filtration, and ion-exchange and affinity chromatography. It was observed to migrate as a single band on one-dimensional sodium dodecyl sulphate--polyacrylamide gels, with a molecular mass of approximately 83 kilodaltons (kDa). On two-dimensional gels it resolved into four polypeptide species with isoelectric points in the acidic range, which on staining with periodic acid--Schiff method were demonstrated to be glycosylated. In the native state, HSP 80 had a molecular size of approximately 610 kDa.     

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Isolation and amino-terminal sequence of a novel cellular growth inhibitor (inhibitory diffusible factor 45) secreted by 3T3 fibroblasts

A growth inhibitory protein named inhibitory diffusible factor 45 (IDF45) has been purified to homogeneity from medium conditioned by dense cultures of mouse 3T3 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fast protein liquid chromatography-purified bioactive material showed a single band corresponding to a protein of 45 kDa. The molecule is a monomer. The pI of the molecule, as determined by isoelectric focusing, is about 6.5. The amino-terminal sequence of the protein was established as Ser-Ala-Gly-Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. The protein inhibits DNA synthesis in chicken embryo fibroblasts in a dose-dependent manner (ED50: 40 ng/ml, approximately 1 nM). The chemical properties of IDF45, i.e. molecular weight and amino-terminal sequence, clearly distinguish it from other known growth inhibitory proteins.     

Characterization of a soluble inorganic pyrophosphatase from Microcystis aeruginosa and preparation of its antibody.

A soluble inorganic pyrophosphatase was isolated from a crude extract of Microcystis aeruginosa by adsorption chromatography. The enzyme was purified to homogeneity as judged by sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis and N-terminal amino acid analysis. The molecular mass was estimated to be 80 kDa by gel filtration chromatography, 87 kDa by nondenaturing polyacrylamide gel electrophoresis, and 28 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 4.5, which is similar to the pI values reported for other soluble inorganic pyrophosphatases. The sequence of 29 N-terminal amino acids was determined; only 4 of these amino acids are identical to those in the sequence of Saccharomyces cerevisiae inorganic pyrophosphatase. M. aeruginosa inorganic pyrophosphatase is a Mg(2+)-dependent enzyme exhibiting a pH optimum of around 7.5. Its KM value for inorganic pyrophosphate was estimated to be 1.30 mM. A specific antibody was raised in chicken to M. aeruginosa inorganic pyrophosphatase. No immunological cross-reactivity was seen when Western blots of partially purified S. cerevisiae or Escherichia coli inorganic pyrophosphatase were probed with the antibody.     

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

Purification and partial characterization of a soluble hemagglutinin from Yersinia pseudotuberculosis

A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.     

Purification, characterization, and partial sequence analysis of a newly identified EF-hand type 13-kDa Ca(2+)-binding protein from smooth muscle and non-muscle tissues

A novel Ca(2+)-binding protein, tentatively designated calgizzarin, has been purified to apparent homogeneity from chicken gizzard smooth muscle by W-7 (N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide))-Sepharose affinity chromatography and ion-exchange chromatography. Application of W-7-Sepharose affinity chromatography to various tissues revealed that calgizzarin-like proteins were abundant in bovine aorta and rabbit lung. Using the same procedure, we could purify a calgizzarin-like protein from rabbit lung. Calgizzarin has a Mr of 13,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 30,000 as determined by gel filtration on a TSK G 3000SW high performance liquid chromatography column, suggesting that calgizzarin seems to be a rodlike protein. The isoelectric point of calgizzarin was found to be pH 5.8. Calgizzarin can exist as a dimer by forming a disulfide bridge. The 45Ca autoradiographic technique showed that the protein binds to Ca2+. On an alkaline/urea gel, calgizzarin migrated faster in the presence of EGTA than in the presence of CaCl2, thereby indicating a Ca(2+)-dependent conformational change in this protein. The partial amino acid sequence (65 amino acid residues) of calgizzarin was seen to be SLLAVFQRYAGREGDNLKLSKKEFRTFMNTELASFTKNQKDPAVVDRMMKRLDINSDGQLDFQEF, and two putative Ca(2+)-binding sites (GREGDNLKLSKKE and D INSDGQLDFQE) were detected. So far as the obtained 65-amino acid sequence is concerned, calgizzarin has approximately a 50% sequence homology with S-100 alpha, 47% with S-100 beta, and 39% with pEL-98 protein.     

Molecular characteristics of beta-galactosidase secreted by Penicillium canescens

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.     

蕲蛇蛇毒中一种新型金属蛋白酶AA-MP-I的纯化和表征

本研究通过嗜硫色谱、Sephadex G-75、蓝胶和POROS HQ20离子交换色谱,从蕲蛇蛇毒中分离得到一种新组分AA-MP-I。该酶为分子量22.9kDa的单体蛋白,等电点为5.55,不含中性糖基,N端序列为STE-FQRYMEIVIVVDHSMVK,结果表明其为新型P-I型金属蛋白酶,对温度敏感,具有抗凝血活性,40℃下抗凝血活性最强,具有出血毒性,无磷脂酶A2活性。