Isolation and properties of the principal liver protein conjugate of a hepatic carcinogen

Sorof, Sam; Bp, Sani; Vm, Kish; Hp, Meloche

doi:10.1021/bi00709a022

Cited by 15 publications

(4 citation statements)

References 23 publications

(28 reference statements)

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“…In one form, the two moieties appear to be noncovalently held in vivo, yet, after protein denaturation, the complex has properties that are compatible with covalent bonding. The properties include withstanding electrophoresis in sodium dodecyl sulfate (11), nonextraction of the bound dye by nonpolar solvents, but solubility in polar solvents after protein degradation (5,6,8). This dual existence of the complex implies that the carcinogen in the native complex is in an activated state that is capable of yielding a reactive electrophile.…”

Section: Discussionmentioning

confidence: 99%

“…When assayed after denaturation and proteolysis, the degraded protein apparently contains covalently bound azo dyes (5,6,8,11). However, speculation has been offered that the native complex may contain activated azocarcinogen (12 The biological turnover of the bound azo dye of the principal liver azoprotein was determined by measurement (see legend, Fig.…”

mentioning

confidence: 99%

“…per subunit) in the isolated mixture of complex and target protein (11), and by the linearity of the plot in Fig. 1 The reversibility of the slowing of the protein-moiety turnover due to carcinogen was examined in experimnirlt 3.…”

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confidence: 99%

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Evidence for a receptor protein of activated carcinogen

Mainigi¹,

Sorof²

1977

Proc. Natl. Acad. Sci. U.S.A.

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During carcinogenesis in rat liver by 3'-methyl-4-dimethylaminoazobenzene, the two moieties of the principal liver carcinogen-protein complex have considerably different turnover rates. With continued ingestion of the azocarcinogen by rats, the bound azo dye in the complex has a half-life of 2.5 ± 0.25 (SD) days, while the protein moiety has a half-life of 8.7 + 1.6 days (probability of identity <0.001). In addition, the interaction of the azocarcinogen with the principal target protein in vivo appears to extend the half-life of the protein itself from 3.3 i 0.2 days in normal liver to 8.7 i 1.6 days (P < 0.001). The slowing of the turnover of the protein by the carcinogen appears to be readily reversible, since soon after the cessation of azocarcinogen feeding, the half-life of the protein returns to that of the target protein in normal liver.The considerable difference in turnover rates of the two moieties of the complex and the reversible effects of the carcinogen in slowing the turnover of the protein moiety suggest that the two moieties of the native azoprotein are noncovalently linked and that they have different biological activities. The native complex appears to contain azo dye in an activated state that is capable of yielding a reactive electrophile, because after protein denaturation the bound azo dye was previously found to have properties that are indicative of covalent linkage to the protein. The retardation in the biological turnover rate of the protein moiety, apparently resulting from interaction with azocarcinogen, is in agreement with the known ligand-induced stabilization in vitro and reduced rate of proteolytic degradation in vivo of other proteins that result from conformational change to a more compact configuration. Our evidence is consistent with the hypothesis that the principal liver carcinogen-protein complex contains hydrophobicalfy bound activated azocarcinogen, whose specificity of reaction with critical macromolecule(s) in nuclei may be directed by the conformationally altered protein of an activated carcinogen-protein complex, i.e., a specific receptor protein containing activated azocarcinogen.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Evidence for a receptor protein of activated carcinogen

Mainigi¹,

Sorof²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The (3)(4)(5)(6). While nonhistone proteins and histones of nuclei have been reported to interact with carcinogens (7-9), none of the principal cytosolic protein targets has been shown thus far to be related to the cell nucleus or chromatin, where chemical carcinogens are generally thought to act in oncogenesis.…”

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confidence: 99%

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

Vinores¹,

Churey²,

Haller³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, shortterm digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500-to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidaseantiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.

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Loss of Regulation of Lipid Metabolism in Morris Hepatomas: A Potential Role for Cytoplasmic Binding Proteins

Mishkin

Halperin

1978

Morris Hepatomas

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Isolation and properties of the principal liver protein conjugate of a hepatic carcinogen

Cited by 15 publications

References 23 publications

Evidence for a receptor protein of activated carcinogen

Evidence for a receptor protein of activated carcinogen

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

Loss of Regulation of Lipid Metabolism in Morris Hepatomas: A Potential Role for Cytoplasmic Binding Proteins

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