Tissue factor (thromboplastin or Factor III), a glycoprotein cofactor, is required for Factor VII to express its catalytic activity, thereby initiating the extrinsic as well as intrinsic pathway of blood coagulation. Human brain tissue factor was purified 2,500-fold to 98% homogeneity from 2% Triton X-100 extraction of acetone dried brain powder with an overall yield of 36%. The method was based upon affinity chromatography utilizing the high affinity binding of tissue factor to Factor VII noncovalently complexed to immobilized anti-Factor VII-agarose beads. The apparent molecular weight of the purified tissue factor is 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point is 4.8-5.1 by column chromatofocussing and flat bed agarose isoelectric focussing.
A variety of stimuli, including bacterial endotoxin, may endow leukocytes with potent procoagulent (tissue factor) activity (TFa). The characterization and purification of this activity was undertaken. Microsomal membrane fractions of endotoxin-stimulated rabbit macrophages were obtained by standard procedure of disruptions and stepwise centrifugations. The membrane fragments containing the highest tissue factor activity were solubilized in sodium barbiturate- deoxycholate buffer (0.05M-0.5%) at pH 8.0 and centrifuged at 180,000 × g for one hour. The concentrated supernatants were subjected to Sephacryl-200 chromatography and eluted with a barbiturate-deoxycholate buffer (0.05M-0.5%). The chromatography dissociated phospholipids from tissue factor apoprotein with apparent loss of activity ensuing. The fractions with high tissue factor activity were pooled and concentrated. Sodium dodecyl sulfate (SDS) was added to the concentrated fraction to a final concentration of 2%. The mixtures were further separated by preparative polyacrylamide gel electrophoresis (prep PAGE). Rabbit brain tissue factor was prepared similarly. Contrasting with rabbit brain preparations, where the TFa eluted as a single peak from prep PAGE, the leukocyte TFa eluted from prep PAGE as 3 or 4 peaks. On analytical PAGE, the different peaks showed slightly different mobilities, the tissue factor activity migrating discretely with each band. A molecular weight of 43,000 was derived for both the fastest band of leukocyte TF and brain TF. These findings suggest different molecular subspecies for rabbit leukocyte TF. (Supported in part by grants from NIH and the Veterans Administration.)
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTUncatalyzed hydrolysis of deoxyuridine, thymidine, and 5-bromodeoxyuridineRobert Shapiro and Sungzong KangCite this: Biochemistry 1969, 8, 5, 1806–1810Publication Date (Print):May 1, 1969Publication History Published online1 May 2002Published inissue 1 May 1969https://pubs.acs.org/doi/10.1021/bi00833a004https://doi.org/10.1021/bi00833a004research-articleACS PublicationsRequest reuse permissionsArticle Views217Altmetric-Citations80LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Molecular orbital electronic structure calculations for twelve polynuclear aromatic hydrocarbons were performed by the samo method. Results indicate that the carcinogenicity of such aromatic hydrocarbons is related to a K-region π-bond order greater than 0.340. There is no correlation with δ-bond order or overall charge density, perhaps accounting for the success of earlier theoretical treatments based on the π-electron model. Exceptions to a simple K-region treatment are discussed in terms of other models for carcinogenic activity.
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