Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.
We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate.
Derivatives of N-glycylglucosylamine have been prepared in preliminary experiments directed towards the synthesis of " glycineamide ribotide " (I) and its formyl derivative (11) , two early nucleotide precursors. Tetra-Oacetyl-P-D-glucopyranosylamine (111) with chloroacetyl chloride gave the N-chloroacetyl derivative (V) . Replacement of the chloro-by an aminogroup and simultaneous deacetylation, by means of ammonia, gave N-glycylfl-D-glucopyranosylamine (VI) which was readily converted into its N-forpyl derivative. An alternative synthesis of the glycyl compounds involves reaction between the amino-sugar and benzyloxycarbonylglycyl chloride or benzyloxycarbonylglycyl ethyl carbonate, followed by removal of protecting groups, IT has been shown by J. M. Buchanan and his collaborators that the uric acid excreted by pigeons is built up from simple substances supplied in the diet. By feeding isotopically labelled compounds and studying the distribution of isotopes in the uric acid formed, it was found that the carbon and nitrogen atoms of the carboxyl, methylene, and aminogroups of glycine were incorporated as a unit into positions 4, 5, and 7 respectively in uric acid. The carbon at positions 2 and 8 originates from formate, and that at position 6 from carbon dioxide.14 The origin of the nitrogen at positions 1, 3, and 9 was not determined conclusively but recent evidence suggests that the atoms at positions 3 and 9 come from the amide-nitrogen of glutamine and that at position 1 from the a-amino-group of aspartic or glutamic acid.5In pigeon-liver extracts hypoxanthine (6-hydroxypurine) is formed rather than uric acid and it is generally considered that this is the normal precursor of uric acid produced by the living bird. It was shown by Greenberg that the hypoxanthine arose through enzymic hydrolysis of inosinic acid and that the ribose-5 phosphate residue in this compound was attached at quite an early stage in its biosynthesis.s-8 Although all the stages in the H formation of inosinic acid from ribose-5 phosphate, glycine, etc., have not yet been clarified, several of the intermediates have now been isolated or detected chr~matographically.~~ ' 2Recent evidence suggests that N-glycy~-~-ribofuranosy~amine-5 phosphate (" glycineamide ribotide ") (I) is formed from glycine, glutamine, and 5-phosphorylribose-1 pyrophosphate (PRPP) in the presence of enzymes in pigeon liver.lO.ll The stages involved in this synthesis are not yet settled, but it appears that glutamine contributes its amide-nitrogen to form the amide-nitrogen of glycineamide ribotide, and that adenosine
Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes, is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system, at the reproduction level, with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore, uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase), responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue.
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