Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase

We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.     

Purification and characterization of human coagulation factor V

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.     

Purification and characterization of ouabain-binding protein in human plasma

Ouabainlike factors are thought to be a kind of important modulators of salt and water metabolism in essential hypertension. We purified the binding-protein of ouabain (OBP) from human plasma. The amino-terminal sequence of OBP from human plasma, (NH2-TLGQPREPQVYTLPPXREEM-), indicated that OBP is the carboxy-terminal fragment (14.4 kDa by SDS-PAGE) from T218 of IgG2 heavy chain and from A221 of the IgG1 heavy chain constant region. Moreover, plasmin-cleaved Fc fragment (pFc) of IgG possessed the ouabain-binding activity by the gel-filtration method of pFc and authentic ouabain mixture, whereas neither intact, aggregate, nor papain-cleaved Fc fragment did. The amino-terminal sequence of pFc was NH2-THTXPPXPAPELLGGPXVFL-, and this sequence corresponded to the T105 to L125 fragment of the IgG1 heavy chain constant region. The growth of cultured THP-1 cells were arrested in the dose-dependent manner by ouabain, which was inhibited by the addition of 20 microg/mL of pFc. These results suggested that plasmin-cleaved Fc of human IgG is one of the binding protein of ouabain/ouabainlike factor(s) in human plasma.     

Purification and characterization of the human interleukin-18 receptor

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.     

Purification and characterization of human sarcomeric mitochondrial creatine kinase

PURPOSE: A dose-escalation study of irinotecan hydrochloride (CPT-11) combined with fixed-dose cisplatin was conducted to determine the maximum-tolerated dose (MTD), dose-limiting toxicities, and objective response rate in patients with advanced gastric cancer. PATIENTS AND METHODS: Twenty-four patients with or without prior chemotherapy were enrolled. All patients were assessable for toxicities and response. On day 1, CPT-11 was administered as a 90-minute intravenous (I.V.) infusion, which was followed 2 hours later by a 120-minute I.V. infusion of cisplatin 80 mg/m2. CPT-11 alone at the same dose was administered again on day 15. The treatment was repeated every 4 weeks until disease progression was observed. The initial dose of CPT-11 was 60 mg/m2, and was escalated in increments of 10 mg/m2 until severe or life-threatening toxicity was observed. RESULTS: The MTD of this combination was CPT-11 80 mg/m2. At this dose level, 16.7% of patients (two of 12) had leukopenia of less than 1,000/microL, 66.7% (eight of 12) had neutropenia of less than 500/microL, and 16.7% (two of 12) had severe diarrhea of grade 4 during the first course. The dose-limiting toxicity was neutropenia. Ten patients achieved a partial response (PR), and the overall response rate was 41.7% among 24 patients (95% confidence interval, 21.9% to 61.4%). CONCLUSION: The recommended dose and schedule is CPT-11 70 mg/m2 on days 1 and 15 and cisplatin 80 mg/m2 on day 1 every 4 weeks. This combination of CPT-11 and cisplatin, considered to be active against advanced gastric cancer with acceptable toxicity, should be further assessed in a phase II study.     

Purification and characterization of angiogenic factor from human bladder cancer

An angiogenic factor from human transitional cell cancer of bladder was purified by protein extraction, cation exchange chromatography, gel filtration high-performance liquid chromatography (GE-HPLC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The purified substance was named as bladder cancer angiogenic factor (BCAF). Biological activity of the BCAF was assessed by using the method of chick embryo chorioallantoic membrane (CAM) assay and 3H-TdR incorporation into DNA in Balb/c 3T3 cells. The BCAF displayed the potent activities of neovascularization in CAM and DNA synthesis in Balb/c 3T3 cells. The ultrastructural features of blood vessels induced by the BCAF were similar to the blood vessels in tumors. The BCAF contained a protein with an approximate molecular weight of 15,000 D, which was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Amino acid compositions of the BCAF were also analysed by acid hydrolysis.     

Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen

L1 is a major and highly immunogenic protein component of human granulocytes. It was purified from random samples of blood-donor leukocytes. An antiserum to L1 was initially raised in rabbits by immunization with protein fractions from preparative agarose gel electrophoresis. The main problem in purifying L1 was its poor stability when carried through multiple steps. Preparative isoelectric focussing was therefore adopted as an efficient one-step method. The isoelectric focussing pattern of L1 was greatly influence by the presence of EDTA or calcium ions in the sample buffer. With low EDTA concentrations or calcium excess, L1 focussed as seven protein bands in two regions, pH 6.1-6.5 and pH 7.6-8.4. Conversely, L1 was found only in the pH-6.1-6.5 region when excess EDTA was added to the sample. Irrespective of conditions, the bulk of L1 focussed at pH 6.3 and pH 6.5. Also the electrophoretic mobility of L1 was strongly influenced by calcium ions, suggesting uptake of calcium by the protein. In the presence of calcium, L1 adhered to glass surfaces and filters used for concentration of protein solutions. The latter problem could be prevented by addition of EDTA. The molecular mass of L1 was determined to be about 36.5 kDa. The molecule was shown to consist of three non-covalently linked 12.5-kDa subunits.     

Purification and characterization of human liver-specific membrane lipoprotein (LSP)

A liver-specific lipoprotein (LSP) which is present in 105,000 g supernatants of human liver has previously been shown to produce a lesion resembling chronic active hepatitis in rabbits immunized with human liver fractions containing this lipoprotein. In addition, it has been implicated as the principal target antigen involved in the liver cell cytotoxicity exhibited in vitro by lymphocytes from patients with chronic active hepatitis. The organ-specificity and species cross-reactivity of LSP is confirmed. Although very labile, is has been prepared in a stable form by gel filtration of Sepharose 6B in a Tris buffer containing 1 mm disodium EDTA. LSP is also stable in a borate buffer containing EDTA but is unstable in a number of other buffer systems tested. When prepared by this method it contains approximately 2% albumin as the only detectable contaminant. Gel filtration studies on the apoprotein of LSP revealed that it has an apparent mol. wt of between 4 X 10(6) and 20 X 10(6). SDA-polyacrylamide gel electrophoresis indicated that the lipoprotein may be composed of up to thirteen sub-units of different molecular sizes.     

Purification and characterization of GDP-L-Fuc: N-acetyl beta-D-glucosaminide alpha1-->6fucosyltransferase from human blood platelets

c-6-L-Fucosyltransferase (alpha1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man alpha1,3 antenna was substituted with GlcNacbeta1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP-L-fucose were 29 and 28 microM, respectively. The optimum pH of the enzyme was 6.0. The activity of alpha1,6FucT was abolished in the presence of beta-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.     

Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.     

Purification and characterization of a human bradykinin binding protein from inflammatory cells

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.     

Purification and characterization of the CA 125 tumor-associated antigen from human ascites

CA 125 is an antigenic determinant associated with epithelial ovarian carcinomas, which is recognized by a monoclonal antibody, OC 125. The biochemical structure, the immunological characteristics and the physiological function of CA 125 are unknown, principally because the molecule expressing it has not been purified to homogeneity. In the present study, we developed a single, one-step method for purifying CA 125 by column affinity chromatography, using the OC 125 antibody as immobilized ligand. The column proved to be highly specific for the purification of CA 125 from human ascites (HA). The antigen that eluted from the column has a specific activity of 6,240 +/- 120 U of CA 125/mg protein, the specific activity in the initial HA samples being 100 +/- 12 U/mg protein. The purified, immunoreactive CA 125 (IR-CA 125) was shown to be proteinaceous in nature. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration characterization showed that the purified antigen exists as a high molecular weight (MW) complex, of up to 1.5 million daltons, which could be dissociated under strong denaturing conditions, giving rise to moieties with an apparent MW of 205 and 55 kD. IR-CA 125 was also associated with a lower MW protein, with an apparent MW of 10-15 kD. The 205-kD MW protein was immunoreactive CA 125, as measured by immunoradiometric assay after being electroeluted from the polyacrylamide gel. Furthermore, when the affinity-purified antigen was subjected to SDS-PAGE, followed by immunoblotting, the lane which was reactive with the iodinated OC 125 antibody gave rise to a band with a molecular mass of 205 kD. Our results suggest that, on an analytical scale, the affinity column is useful for the purification of CA 125. The purified antigen is being used to investigate the possible role of CA 125 in the growth, development and physiological characteristics of human ovarian carcinomas in in vitro studies.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase (M(r) 31,000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 degrees C, respectively. pH stability ranged from 5 to 9.5 and the t1/2 at 50 degrees C was 490 min. It had a Km of 4.88 mg ml-1 and a Vmax of 588 mumol min-1 mg-1. The activity was inhibited (95%) by AlCl3 (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Purification and characterization of a microsomal bile acid beta-glucosidase from human liver

A human liver microsomal beta-glucosidase has been purified to apparent homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis where a single protein band of Mr 100,000 was obtained under reducing conditions. The enzyme was enriched about 73, 000-fold over starting microsomal membranes by polyethylene glycol fractionation, anion exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by affinity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhibition of activity. The enzyme catalyzed the hydrolysis of 3beta-D-glucosido-lithocholic and 3beta-D-glucosido-chenodeoxycholic acids with high affinity (Km, 1.7 and 6.2 microM, respectively) and of the beta-D-glucoside (Km, 210 microM) and the beta-D-galactoside of 4-methylumbelliferone. The ratio of relative reaction rates for these substrates was about 6:3:11:1. No activity was detectable toward 6beta-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta-D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitation studies using antibodies prepared against lysosomal glucocerebrosidase showed no cross-reactivity with microsomal beta-glucosidase suggesting that these two enzymes are antigenically unrelated.     

Purification and characterization of endothelin-1 degradation activity from porcine kidney

In order to identify the membrane-bound peptidase that is responsible for the degradation of endothelin (ET), an endothelin-1 (ET-1) degradation enzyme was solubilized from membrane fractions of porcine kidney with 1% Triton X-100, and subsequently purified by column chromatographies, i.e., diethylamino-Sepharose ion exchange, gel permeation, Con A Sepharose and hydroxyapatite chromatography. On DEAE-Toyopearl ion exchange column chromatography, the ET degradation enzyme and aminopeptidase were separated, but ET degradation enkephalinase activities were not separable. In order to separate ET degradation enzyme and enkephalinase, the active fractions were loaded on each of the column chromatographies: sephacryl S-200, Con A Sepharose or hydroxyapatite. The ET degradation activities were co-migrated with enkephalinase activities on all of the three chromatographies. In addition, the ET degradation activities were inhibited by thiorphan, phosphoramidon and EDTA, which are known to inhibit enkephalinase. These results suggest that ET degradation activity in the membrane fractions of the kidney is related to enkephalinase and may be involved in the degradation of ET-1 in vivo.     

Purification and characterization of virginiamycin M1 reductase from Streptomyces virginiae

Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrocyclic lactone antibiotic belonging to the virginiamycin A group. S. virginiae possesses an activity which stereospecifically reduces a 16-carbonyl group of VM1, resulting in antibiotically inactive 16R-dihydroVM1. The corresponding VM1 reductase was purified to homogeneity from crude extracts of S. virginiae in five steps, with 5,650-fold purification and 23% overall yield. The N-terminal amino acid sequence was determined to be MAIKLVIA. The purified enzyme showed an apparent Mr of 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 280,000 by native molecular sieve high-performance liquid chromatography, indicating the tetrameric nature of the native enzyme. NADPH served as a coenzyme for the reduction, with a Km value of 0.13 mM, but NADH did not support the reaction, even at a concentration of 5 mM, indicating the NADPH-specific nature of the enzyme. The Km for VM1 was determined to be 1.5 mM in the presence of 2 mM NADPH. In the reverse reaction, only 16R-dihydroVM1, not the 16S-epimer, served as a substrate, with a less than 0.1% overall reaction rate compared to that of the forward reaction, confirming that the VM1 reductase participates solely in VM1 inactivation in vivo.     

Purification and characterization of pili of a Vibrio cholerae non-O1 strain

Pili of the Vibrio cholerae non-O1 strain V10 were purified and characterized. The V10 pili were physicochemically and immunologically different from those of the previously reported V. cholerae non-O1 strain S7, although the pili of the two strains had homologous N-terminal amino acid sequences. V10 plus antigen was detected only in V. cholerae non-O1 strains.