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Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS)