Purification and primary structure of a macromolecular-translocation inhibitor II of glucocorticoid-receptor binding to nuclei from rat liver. Inhibitor II is the 11.5-kDa Zn2+-binding protein (parathymosin).
The nuclear binding of the activated glucocorticoid-receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI-I, MTI-II and MTI-III). In this study, we purified the most potent of the three species (MTI-II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two-dimensional PAGE. Purified MTI-II inhibits GR binding to DNA containing glucocorticoid-response elements. To obtain the amino acid sequence of MTI-II, we digested the MTI-II with endopeptidases. The N-terminal amino acid sequences of the four digested fragments indicated that MTI-II is an 11.5-kDa Zn2+-binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI-II (ZnBP, parathymosin) and that GR binds to the MTI-II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.
Okamoto
K
K
Department of Biochemistry, St. Marianna University School of Medicine, Kanagawa, Japan.
Isohashi
F
F
D000315:Adrenalectomy; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D007526:Isoelectric Point; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D013947:Thymosin; D004247:DNA; C044498:parathymosin alpha; D015032:Zinc