Summary
A nuclear antigen was purified from the 0.01 M Tris-HCl/pH 8 extract of nuclei of the Burkitt tumor Namalwa cell line to electrophoretic homogeneity by DEAE cellulose chromatography, affinity chromatography, and preparative isoelectric focusing. The yield of antigens was 0.02% of the nuclear 0.01 M Tris-HCl/pH 8 extract. On two-dimensional gel electrophoresis, the major antigen separated into two adjacent protein spots with molecular weights of 68,000 and an approximate pI of 6.3 (68/6.3 A and 68/6.3 B). A minor antigen had a molecular weight of 61,000 and pI of 6.0 68/6.0). Fourteen 125I-labeled peptides were obtained from the tryptic digest of the major antigen (68/6.3 A and 68/6.3 B). The amino-acid composition analysis of the purified antigens indicated that the amino acids in the highest content were glycine (15%), glutamic acid (11.6%), and serine (9%); the ratio of acidic to basic amino acids was 1.95. In studies on nucleolytic activity, the purified antigen produced a single-stranded and then a double-stranded cleavage of PM 2 and pBR 322 DNA. This antigen is the first purified nuclear antigen that reacts with the HeLa-specific nucleolar antibodies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- PBS:
-
8.5mM Na2HPO4/1.6mM NaH2PO4/0.145 M NaCl/pH 7.2
- BSA:
-
bovine serum albumin
- PMSF:
-
phenylmethylsulfonlyfluoride
- IF gel:
-
isoelectric focusing gel
References
Attardi DG, DePaolis A, Tocchini-Valentini GP (1981) Purification and characterization of Xenopus laevis type I topoisomerase. J Biol Chem 256:3654–3661
Baldi MI, Benedetti P, Mattoccia E, Tocchini-Valentini GP (1980) In vitro catenation and decatenation of DNA and a novel encaryotic ATP-dependent topoisomerase. Cell 20:461–467
Bradford M (1976) A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–252
Busch RK, Busch H (1977) Antigenic proteins of nucleolar chromatin, of Novikoff hepatoma ascites cells. Tumori 63:347–357
Busch H, Busch RK, Chan PK, Daskal Y, Gyorkey F, Gyorkey P, Kobayashi M, Smetana K, Sudhakar S (1980a) A nucleolar antigen in human malignant tumors Transplant Proc 12:99–102
Busch H, Busch RK, Chan PK, Gyorkey F, Smetana K (1980b) The human tumor nucleolar antigen. Trends Pharmacol Sci 1:436–439
Busch H, Daskal Y, Gyorkey F, Smetana K (1979a) Silver staining of nucleolar granules in tumor cells. Cancer Res 39:857–863
Busch H, Gyorkey F, Busch RK, Davis FM, Gyorkey P, Smetana K (1979b) A nucleolar antigen found in a broad range of human malignant tumor specimens. Cancer Res 39:3024–3030
Cann JR (1979) Electrophoresis and isoelectric focusing of interacting systems. In: Electrokinetic Separation Methods. Elsevier/North-Holland Biomedical Press, pp 369–387
Campoux JJ, McConaughy BL (1976) Purification and characterization of the DNA untwisting enzyme from rat liver. Biochemistry 15:4638–4642
Chan PK, Feyerabend A, Busch RK, Busch H (1980) Identification and partial purification of human tumor nucleolar antigen 54/6.3 Cancer Res 40:3194–3201
Collett MS, Erikson E, Erikson RL (1979) Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol 29:770–781
Crumplin GC (1981) The involvement of DNA topoisomerases in DNA repair and mutagenesis. Carcinogesis 2:157–160
Culp LA, Brown GM (1970) RNA methylases from rat liver nucleoli. Arch Biochem Biophys 137:222–230
Davis FM, Gyorkey F, Busch RK, Busch H (1979) Nucleolar antigen found in several human tumors but not in the nontumor tissues studies. Proc Natl Acad Sci USA 76:892–896
Fraker PJ, Speck, Jr. JC (1978) Protein and cell membrane iodination with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun 80:849–857
Gravery JS, Cremer NE, Sussdorf DH (1977). In: Methods in Immunology, pp 218–226, W.A. Benjamin, Ma USA
Hirs CHW (1976). In: Methods in Enzymology, Vol 11. Academic Press, New York, pp 199–203
Huang, CH, Mirabelli CK, Jan Y, Crooke ST (1981). Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues. Biochemistry 20:233–238
Lischwe MA, Smetana K, Olson MOJ, Busch H (1979). Protein C 23 and B 23 are the major nucleolar silver staining proteins. Life Sci 25:701–708
Liu LF, Miller KG (1981). Eukaryotic DNA topoisomerases. Two forms of type I DNA topoisomerases from HeLa cellnuclei Proc Natl Acad Sci USA 78:3487–3491
Marashi F, Davis FM, Busch RK, Savage HE, Busch H (1979) Purification and partial characterization of nucleolar antigen-1 of the Novikoff hepatoma. Cancer Res 39:59–66
Purchio AF Erikson E, Brugge JS, Erikson RL (1978). Identification, of a polypeptide encoded by the avian sarcoma virus src gene. Proc Natl Acad Sci USA 75:1567–1571
Smetana K, Busch RK, Hermansky F Busch H (1979). Nucleolar immunofluorescence in human hetalogical malignancies. Life Sci 25:227–234
Takami H, Busch H (1979). Two-dimensional gel electrophoretic comparison of proteins of nuclear fractions of normal liver and Novikoff hepatoma. Cancer Res 39:507–518
Taylor CW, Yeoman LC, Woolf LM, Busch H (1979). Isolation of a 70,000 molecular weight antigen of the Novikoff hepatoma. Biochemistry 18:4049–4054
Tse Y-C, Wang JC (1980) E. coli and M. luteus DNA topoisomerase I can catalyze catenation or decatenation of double stranded, DNA rings. Cell 22:269–276
Yeoman LC, Jordan JJ, Busch RK, Taylor CW, Savage H, Busch H (1976). A fetal protein in chromatin of Novikoff hepatoma and Walker 256 carcinosarcoma tumors that is absent from normal and regenerating rat liver Proc Natl Acad Sci USA 73:3258–3262
Author information
Authors and Affiliations
Additional information
These studies were supported in part by the Human Tumor Nucleolar Antigen Grant, CA-27534, The Farish Fund, the Pauline Sterne Wolff Memorial Foundation, the Bristol-Myers Fund, and the Cancer Research Program Grant CA-10893, awarded by the National Cancer Institute, DHEW
Rights and permissions
About this article
Cite this article
Chan, PK., Frakes, R.L., Busch, R.K. et al. Isolation and partial characterization of a nuclear antigen (68/6.3) from the Namalwa cell line (a Burkitt lymphoma). J Cancer Res Clin Oncol 103, 7–16 (1982). https://doi.org/10.1007/BF00410301
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00410301