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Title: Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

Abstract

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate bindingmore » region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

Authors:
; ;
Publication Date:
Research Org.:
Univ. of Calgary, Alberta
OSTI Identifier:
6127431
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 26:11
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GLUCOPROTEINS; MOLECULAR STRUCTURE; HYALURONIC ACID; BIOCHEMISTRY; LABELLED COMPOUNDS; BIOCHEMICAL REACTION KINETICS; ELECTROPHORESIS; FIBROBLASTS; METHIONINE; MICE; MOLECULAR WEIGHT; ONCOGENIC VIRUSES; SULFUR 35; AMINES; AMINO ACIDS; ANIMAL CELLS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBOHYDRATES; CARBOXYLIC ACIDS; CHEMISTRY; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DRUGS; EVEN-ODD NUCLEI; ISOTOPES; KINETICS; LIGHT NUCLEI; LIPOTROPIC FACTORS; MAMMALS; MICROORGANISMS; MUCOPOLYSACCHARIDES; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PARASITES; POLYSACCHARIDES; PROTEINS; RADIOISOTOPES; REACTION KINETICS; RODENTS; SACCHARIDES; SOMATIC CELLS; SULFUR ISOTOPES; VERTEBRATES; VIRUSES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Turley, E.A., Moore, D., and Hayden, L.J.. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells. United States: N. p., 1987. Web. doi:10.1021/bi00385a007.
Turley, E.A., Moore, D., & Hayden, L.J.. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells. United States. doi:10.1021/bi00385a007.
Turley, E.A., Moore, D., and Hayden, L.J.. Tue . "Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells". United States. doi:10.1021/bi00385a007.
@article{osti_6127431,
title = {Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells},
author = {Turley, E.A. and Moore, D. and Hayden, L.J.},
abstractNote = {A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.},
doi = {10.1021/bi00385a007},
journal = {Biochemistry; (United States)},
number = ,
volume = 26:11,
place = {United States},
year = {Tue Jun 02 00:00:00 EDT 1987},
month = {Tue Jun 02 00:00:00 EDT 1987}
}
  • Cell lines from AKR and BALB/c mouse embryos were compared for their sensitivity to x-ray induction of endogenous type C virus. K-Balb cells, a Balb/3T3 cell line nonproductively transformed by Kirsten murine sarcoma virus, were found to be sensitive to s-irradiation. At a dose as low as 50 R, x-rays induced virus expression in K-Balb cells, and the induction frequency increased with increasing dose of x-rays up to 400 R. Among two classes of inducible endogenous viruses carried by K-Balb cells, only Balb:virus-2 was activated by x-irradiation, whereas both Balb:virus-1 and Balb:virus-2 were activated after the cells were treated withmore » 5-iodo-2'-deoxyuridine. UV light and 4-nitroquinoline 1-oxide were also shown to induce virus expression in K-Balb cells.« less
  • Cell lines from AKR and BALB/c mouse embryos were compared for their sensitivity to x-ray induction of endogenous type C virus. K-Balb cells, a Balb/3T3 cell line nonproductively transformed by Kirsten murine sarcoma virus, were found to be sensitive to x-irradiation. At a dose as low as 50 R, x rays induced virus expression in K-Balb cells, and the induction frequency increased with increasing dose of x rays up to 400 R. Among two classes of inducible endogenous viruses carried by K-Balb cells, only Balb:virus-2 was activated by x irradiation, whereas both Balb:virus-1 and Balb:virus-2 were activated after the cellsmore » were treated with 5-iodo-2'-deoxyuridine. uv light and 4-nitroquinoline 1-oxide were also shown to induce virus expression in K-Balb cells. The virus-induction frequency for these physical and chemical carcinogens was much lower (approx. = 3 x 10/sup -4/) than that for 5-bromo-2'-deoxyuridine (approx. = 1 x 10/sup -1/).« less
  • Transformation of NIH 3T3 cells with Kirsten sarcoma virus (Ki-SV) increased phosphatidylinositol (PI) metabolism. This suggests possible alterations in the phospholipase C (PLC) and PIP/sub 2/-phosphodiesterase (PIP/sub 2/-PDE) activities responsible for hydrolysis of PI and PIP/sub 2/ with Ki-SV transformation. An in vitro assay is employed to study the hydrolysis of exogenously added (/sup 3/H)PI and (/sup 3/H)PIP/sub 2/ with membranes prepared from normal and Ki-SV transformed cells. Association of these activities with membranes appears to be differentially mediated by metals (Ca/sup 2 +/) since chelator treatment dissociates PLC from the particulate fraction. Hydrolysis of PIP/sub 2/ is markedly enhancedmore » (10 fold) by introducing (/sup 3/H)PIP/sub 2/ to membrane preparations in vesicles prepared with excess phosphatidylethanolamine. These activities are dependent on Ca/sup 2 +/ and exhibit a progressive increase in activity between 10/sup -7/M and 10/sup -3/M Ca/sup 2 +/. The optimal pH for PIP/sub 2/-PDE is 7.0, whereas PI specific PLC exhibits optimal activity at pH 5.5. With this in vitro assay system it is possible to demonstrate that GTP-..gamma..-S addition to isolated membranes stimulates PIP/sub 2/-PDE to hydrolyze exogenously added (/sup 3/H)PIP/sub 2/. This should allow direct studies to determine possible differences in GTP-dependent regulation of PI and PIP/sub 2/ hydrolysis with membranes prepared from normal and transformed cells.« less
  • BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were stimated by the method of fiber-autoradiography and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcome virus-transformed BALB/c 3T3 cells.
  • Oncogenes have previously been reported in the DNAs of mouse fibroblast lines which had become transformed after in vitro exposure to the carcinogen 3-methylcholanthrene. These oncogenes are now shown to be versions of the cellular Kirsten ras gene and are therefore homologous to oncogenes detected in a variety of human tumor DNAs.