Purification and photoaffinity labelling of lipid methyltransferase from rat liver.
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ABSTRACT:
SUBMITTER: Pajares MA
PROVIDER: S-EPMC1144264 | biostudies-other | 1984 Oct
REPOSITORIES: biostudies-other
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Project description:Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.
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Project description:Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.
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Project description:A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.
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Project description:In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.
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Project description:Photoaffinity labelling of brush border membrane vesicles from rabbit ileum with radiolabelled 3,3-azo and 7,7-azo derivatives of taurocholate identified integral membrane proteins of molecular masses 93 and 46 kDa, as well as a 14 kDa peripheral membrane protein, as components of the ileal Na+/bile acid transport system [Kramer, Girbig, Gutjahr, Kowalewski, Jouvenal, Müller, Tripier and Wess (1993) J. Biol. Chem. 268, 18035-18046]. Differential photoaffinity labelling in the presence of non-radiolabelled bile acid derivatives led, as expected, to a concentration-dependent decrease in the extent of labelling of the 93 and 46 kDa transmembrane proteins, which are the monomeric and dimeric forms of the ileal bile acid transporter protein. The extent of labelling of the 14 kDa ileal lipid-binding protein (ILBP), however, increased on the addition of unlabelled bile acids, the increase being dependent on the structure of the bile acid added. The possibility of artifacts was excluded by photoaffinity labelling experiments in the frozen state as well as by model calculations. The experimental results suggest that the binding of bile acids to ILBP can increase the affinity of ILBP for bile acids. These results would be in accordance with a substrate-load modification of transport activity and a positive-feedback regulation mechanism for active uptake of bile acid in the ileum.
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