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 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 characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues

The monoclonal antibody UN1 was previously produced in our laboratory on the basis of selective reactivity with human thymocytes and has been classified as unclustered by the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. The antigen recognized by mAb UN1 was found to be expressed on the cell surface of immature human thymocytes, a subpopulation of peripheral T lymphocytes and on several fetal tissues including thymus. The UN1 antigen is purified from children's thymus by ion-exchange and affinity chromatography. Two-dimensional electrophoresis shows that the purified antigen displays microheterogeneity appearing as multiple spots over a pI range 4.4-5.0 at 100-120 kDa. Treatment with neuraminidase results in a retarded migration in SDS-PAGE, an increase in isoelectric point and a reduction in carbohydrate content, indicating a substantial content of sialic acid. Glycosidase digestion and lectin-binding analysis indicate that the carbohydrate residues are essentially O-linked. A preliminary analysis has detected the UN1 antigen in human breast carcinoma tissues but not in normal breast. The biochemical features and the pattern of expression of the UN1 antigen indicate that this molecule may have the characteristics typical of the family of cell-membrane-associated mucin-like glycoproteins; a number of these molecules are thought to have a role in cell-cell interaction, tumor progression and metastasis.     

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 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 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 a DNA binding protein in a nuclear scaffold fraction from rat ascites hepatoma cells

Our most recent work [Hibino et al. (1995) Cancer Lett., 88, 49-55] has shown that the selective binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat ascites hepatoma cells (P230) depend on the degree of sequence-directed bending of the helix axis. In the present experiment, this protein has been highly purified and isolated by a series of column chromatographic procedures to migrate as a single band to a molecular weight position of 230 kd on a SDS-polyacrylamide gel. A filter binding assay showed that the binding of a repetitive AT-rich component (369 bp XmnI fragment) from the hepatoma nucleus, which has a strongly bent overall structure, to isolated P230 is based on a cooperative mode of interaction. Distamycin A, which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding to this protein. These results suggest that AT-rich regions in highly repetitive DNA cause bending of the helix axis to be recognized by nuclear scaffold protein(s). Moreover, it has been shown that the nuclear scaffold fraction from rat liver or an actively growing hepatocyte cell line (Ac2F cells) does not contain P230, but does have a repetitive bent DNA binding protein (P130), which has an apparent molecular weight of 130 kd. In addition, the immunoblot analysis showed that mouse anti-P130 antiserum reacts with P230. Thus, the results in the present study imply that there is some difference in the higher order structure of the nuclear DNA attachment region between rat liver or actively growing hepatocytes and the hepatoma, although P230 appears to be immunochemically similar to P130.     

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 properties of RNA polymerase I from Ehrlich ascites cells

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.     

Purification and characterization of high molecular weight hCG from human first trimester placenta

Human chorionic gonadotropin (hCG) in first trimester placental cells is composed of immature alpha- and beta-subunits containing only N-linked high-mannose sugar chains. Intracellular immature intermediates are accumulated in rough endoplasmic reticulum in much greater quantity than mature hCG composed of mature subunits. We have previously shown that this immature hCG might be bound to other protein(s), including an ATP-binding protein, forming high molecular weight-hCG (HMW-hCG), which is not aggregate of immature hCG alone. To identify the ATP-binding protein forming the HMW-hCG in detail, proteins in HMW-hCG preparation were photoaffinity-labeled with 8-azido-[alpha-32P]ATP. Autoradiography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the labeled protein with M(r) = 78000 was immunoprecipitated with any antibody against alpha-subunit, beta-subunit and hCG, indicating that this protein is bound to immature hCG. Furthermore, to determine whether some other proteins associate to form HMW-hCG, we purified HMW-hCG without breakdown to its components using columns of DE52, Heparin-Sepharose and Sephacryl S-300. As the final step of the purification, HMW-hCG was allowed to adsorb on a column of ATP-agarose and anti-hCG IgG-agarose, respectively. SDS-PAGE analysis of eluted proteins from the columns bound to the respective column via the constituent of HMW-hCG, such as ATP-binding protein or immature hCG, showed four common protein bands with molecular weights of 78000, 43000, 28000 and 20000. The protein with M(r) = 43000 was stained with any antibody against alpha-subunit, beta-subunit and hCG, indicating it to be immature hCG. The protein band with M(r) = 78000, which might correspond to the ATP-binding protein described above, was stained with anti-heat shock protein 70 (HSP70) monoclonal antibody. To confirm the association of immature hCG and HSP70-like protein, immature hCG preparation was incubated with HSP70-like protein purified from placental extracts. The molecular weight change of immature hCG appeared to increase by this incubation and was close to HMW-hCG, but not exactly the same. These results suggest that immature hCG intermediate exists as HMW-hCG containing HSP70-like protein, which has ATP-binding capacity, and two other proteins in first trimester placental cells.     

Purification and characterization of the D-hydantoinase from Bacillus circulans

A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans. Purification of two hundred forty-three-fold was achieved with an overall yield of 12%. The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000. This enzyme is an acidic protein with an isoelectric point of 4.55. The enzyme is sensitive to thiol reagent and requires metal ions for its activity. The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C. The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS. These remarkable properties are used for the purification procedure.     

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.     

CA125-producing adenocarcinoma of the seminal vesicle

A case of primary seminal vesicle carcinoma is reported. The tumor was a CA125-producing adenocarcinoma consisting of fine papillary-tubular, intricate branching or anastomosing glandular structures and was composed of small cuboidal, but occasionally hobnailed, cells with mostly clear, but occasionally granular, cytoplasm. Some tumor cells showed evidence of secretion of seromucinous materials into the interpapillary and cystic space. Immunohistochemically, almost half of the tumor cells expressed a positive reaction with anti-CA125, a common serological marker for ovarian epithelial carcinomas; however, no tumor cells expressed any other serological tumor markers such as carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, prostatic specific acid phosphatase, or prostatic specific antigen. The patient showed a high level of serological CA125, which fluctuated parallel with the growth, removal and recurrence of the tumor. The morphological and immunohistochemical findings suggested a close relationship between the present tumor and clear cell carcinoma of the ovary, which is thought to be of a Müllerian-Wolfian duct origin.     

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.     

Characterization of peptides that bind the tumor-associated Thomsen-Friedenreich antigen selected from bacteriophage display libraries

Peptides with high affinities and specificities for numerous proteins and nucleic acids have been previously identified from random peptide bacteriophage display libraries. Here, random peptide bacteriophage display libraries were used to identify sequences that bound the cancer-associated Thomsen-Friedenreich glycoantigen (T antigen). The T antigen, present on most malignant cells, contains an immunodominant Gal beta1 --> 3GalNAc alpha disaccharide unmasked on the surfaces of most carcinomas. This antigen has been postulated to be involved in tumor cell aggregation and metastasis. Two 15 amino acid random peptide bacteriophage display libraries were affinity selected with glycoproteins displaying T antigen on their surfaces. Sequence analysis revealed that many of the peptides shared homology with sugar recognition sites in several carbohydrate-binding proteins. A comparison of affinity selected sequences from both libraries yielded a common motif (W-Y-A-W/F-S-P) rich in aromatic amino acids. Four peptides, corresponding to the affinity selected sequences, were chemically synthesized and characterized for their carbohydrate recognition properties. The synthetic peptides exhibited high specificities and affinities to T antigen displayed on asialofetuin or conjugated to bovine serum albumin (Kd = 5 nM for MAP-P30 binding to asialofetuin) as well as free T-antigen disaccharide in solution (Kd = 10 microM for MAP-P30, 20 microM for P10). Two peptides, P30 and P10, demonstrated high affinities and specificities for both asialofetuin and T antigen in solution. Iodination of a lone tyrosine residue in each sequence dramatically reduced their abilities to bind T antigen, suggesting that the tyrosine residue plays an important role in carbohydrate recognition. That these peptides are of functional significance is evidenced by the ability of both P30 and P10 to inhibit asialofetuin-mediated melanoma cell aggregation in vitro and to compete with peanut lectin for binding to T antigen displayed on the surface of MDA-MB-435 breast carcinoma cells in situ.