A cellular 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line. Purification and characterization

A cellular binding protein for 3,3',5-triiodo-L-thyronine (T3) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from A431 human epidermoid carcinoma cells. The binding activity is T3 specific. Analysis of the equilibrium binding data indicated that the binding protein has one class of binding sites for T3 with a Kd of (17 +/- 3) nM and Bmax of (1.8 +/- 0.6) pmol/50 micrograms of protein. The pH optimum for binding is 6.8. The T3 binding protein elutes from Sephadex G-200 in an included peak which has a Stokes radius of 40 A and sediments on glycerol gradients at 3.7 S. By affinity labeling with [3,5-125I]thyroxine a protein with a molecular weight of 58,000 was specifically labeled. Its isoelectric point was determined to be 7.1, which is different from the reported pIs of other thyroid hormone binding proteins. p58 was successively purified to apparent homogeneity by chromatography on Sephadex G-200, QAE-Sephadex, SP-Sephadex, and hydroxylapatite. Approximately 50 micrograms of purified protein was obtained from 2.5 X 10(9) cells with a yield of 1.1%. The purified protein retains its binding activity. The specific binding activity is enriched by approximately 1000-fold. With the availability of a purified protein with T3 binding activity, it becomes possible to study its cellular function.     

A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies

We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.     

Molecular cloning of the gene coding for the human T cell differentiation antigen CD7

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.     

Thyroid hormone down-regulates p55, a thyroid hormone-binding protein that is homologous to protein disulfide isomerase and the beta-subunit of prolyl-4-hydroxylase

We have previously characterized a cellular thyroid hormone-binding protein (p55) that is found concentrated on the lumenal face of the endoplasmic reticulum and nuclear envelope (Cheng, S.-y., Hasumura, S., Willingham, M.C., and Pastan, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 947-951). To understand the role p55 plays in thyroid hormone action, we examined the regulation of p55 by 3,3',5-triiodo-L-thyronine (T3). Rat pituitary tumor GH3 cells cultured in regular medium, thyroid hormone-depleted medium (Td medium), or Td medium supplemented with 50 nM T3 (Td + T3 medium) were metabolically labeled with [35S]methionine and immunoprecipitated with antibodies against p55. Treatment with T3 caused a fall in p55 levels. Poly(A+) RNA from cells cultured in regular, Td, or Td + T3 medium was hybridized to a cDNA from p55. T3 withdrawal or addition had no effect on p55 mRNA levels. Furthermore, the initial rates of synthesis of p55 from cells cultured in regular, Td, and Td + T3 were found to be similar. However, analysis of the decay curves from cells in which p55 was pulse-labeled with [35S]methionine indicated that p55 is 2-fold less stable in T3 containing medium. These results indicated that down-regulation of p55 by T3 occurs at the post-translational level. Since DNA sequence analysis indicates that p55 is identical to protein disulfide isomerase and the beta-subunit of prolyl-4-hydroxylase, T3 may mediate its effects on the synthesis, secretion, and/or transport of proteins via p55.     

Acute reversible cataract due to nitrocompounds in Japanese quail (Coturnix coturnix japonica)

The present study was designed to examine the usefulness of the Japanese quail as an experimental model of cataractogenic activity. Chemicals, 2, 6-dibromo-4-nitro-phenol (2, 6-D), 2, 4-dinitroanisole (2, 4-DA), and 2, 4-dinitrophenol (2, 4-D; for the positive control), were administered singly through an oral route to 2-week old male Japanese quails to investigate the reversibility of cataracts. A single administration of 2, 4-D (36 and 43 mg/kg) produced reversible cataract in 14 of 16 animals (87.5%). This cataract was seen 1 or 2 hours after treatment and continued for 1 to 12 hours. Treatment with 2, 6-D (20 and 25 mg/kg) and 2, 4-DA (120 and 150 mg/kg) caused cataracts in 7 of 11 (63.6%) and 8 of 8 surviving animals (100%), respectively. Cataracts produced by 2, 6-D and 2, 4-DA, which were observed from 1 and 2 to 4 hours after the treatment, continued for 6 to 15 and 1 to 13 hours, respectively. Mortalities in the 25 mg/kg group of 2, 6-D, 120 mg/kg and 150 mg/kg group of 2, 4-DA were found in 2 of 5 animals, 1 of 5 animals and 5 of 9 animals, respectively. These results indicate that the Japanese quail is useful as an animal model to evaluate toxicity to the eye and cataractogenesis.     

Properties of the raw-starch digesting amylase of Aspergillus sp. K-27: a synergistic action of glucoamylase and alpha-amylase

Glucoamylase and alpha-amylase have been purified from a crude enzyme preparation of Aspergillus sp. K-27. The former was thermostable and seemed to have a "starch-binding site", judging from the results of a kinetic study, and the latter synergistically enhanced the degradation of starch granules with glucoamylase.     

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration.

To understand the regulation by thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M2) during liver regeneration, the synthesis of p58-M2 was evaluated. The synthesis of p58-M2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is approximately 25% higher in hyper- than in hypothyroid rats. However, T3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T3 increased the synthesis of p58-M2 by approximately 2.5-fold. During regeneration, however, the change in the synthesis of p58-M2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M2 remained virtually unchanged during regeneration. These results indicate that T3 regulates the synthesis of p58-M2 in mature and regenerating liver.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

Mature ferredoxin-NADP reductase with a glutaminyl residue at N-terminus from spinach chloroplasts

When 35%-acetone extract of spinach chloroplasts was separated by SDS-PAGE, ferredoxin-NADP reductase (FNR) appeared as a single band at a molecular mass of 35 kDa. After the polypeptides on the SDS-PAGE plate were electroblotted onto PVDF membrane, the FNR band was cut out and analyzed for N-terminal structure in a gas-phase protein sequencer. Two different FNR peptides were identified: one with glutamine at its N-terminus (Gln-FNR) and the other with -pyroglutamic acid (tFNR) fraction was extracted from chloroplasts with their loosely bound FNR (lFNR) fraction removed in advance. The tFNR fraction contained Gln-FNR only. The Gln-FNR could be highly purified by affinity chromatography using a ferredoxin column. The purified Gln-FNR was digested with arginyl endopeptidase for peptide mapping and partial sequence analysis. Primary structure of Gln-FNR differed from that of lFNR loosely bound FNR - tFNR tightly bound FNR - -pyroglutamic acid at N-terminus