pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

白兰瓜果实输入~(14)C-葡萄糖的转化与蔗糖合成

~(14)C 追踪试验结果表明,白兰瓜幼果中输入的~(14)C-葡萄糖,50%以上转化为稀酸水解和稀酸不水解的结构物质;果实发育后期,输入后48小时,在果肉和种子中分别只有18%和32%的~(14)C 参入结构物质。根据醇溶性糖的纸层析鉴定,幼果薄片渗入的~(14)C-葡萄糖仅转化为果糖,而发育后期果实则更多转化为蔗糖。显然,幼果的代谢模式是使物质和能量导向结构物质的形成;而后期果实生长已基本停止,物质代谢的方向又转向蔗糖合成的轨道上来。蔗糖合成底物试验结果表明,供给幼果不同底物都只有很低的蔗糖合成活性;发育后期果实供给UDPG+F-6-P 底物时可测出较高的蔗糖合成活性,初步推测白兰瓜中蔗糖合成主要是通过蔗糖磷酸酯合成酶来实现的。     

异大茴香脑新资源——齿叶黄皮的研究

齿叶黄皮是生长于广东省北部石灰岩山地的一种芸香科常绿小乔木,其叶富含精油,枝叶经水蒸汽蒸馏出油率为0.7%。精油经毛细管气相色谱、气相色谱/质谱联用、红外吸收光谱、核磁共振波谱等方法分析,确证含异大茴香脑(isoanethole)93.10%。异大茴香脑对霉菌有较强抑菌活性,并可通过异构化一步转变为大茴香脑。     

Formation of lymphocyte colonies under serum-free culture conditions in normal individuals and patients with chronic lymphocytic leukemia

This paper describes a culture system which supports the formation of B cell and some T cell colonies under serum-free conditions in peripheral blood samples of normal individuals and patients with chronic lymphocytic leukemia (CLL) of B cell type. In this system, serum is replaced by bovine serum albumin, transferrin, cholesterol, insulin and catalase or horseradish peroxidase. In addition, it is necessary to add staphylococcus protein A, mitomycin-treated T cells as feeders and phytohemagglutinin leukocyte-conditioned medium as a source of growth factors. The plating efficiency is greatly enhanced when normal cells are incubated with galactose oxidase prior to plating and when CLL cells are exposed sequentially to neuraminidase and galactose oxidase.     

An immunocytochemical method for the localization of fibronectin in Araldite-embedded specimens

Summary The detection of fibronectin (FN) in osmium-fixed and Araldite-embedded frog skin fragments was studied using a modification of Baskin's procedure (Baskin et al. 1979). Following the removal of Araldite from the semi-thin sections (0.5–1.0 m) with ethanol-NaOH solution, the sections were bleached with hydrogen peroxide. FN was detected by indirect immunoperoxidase method. For precise localization of FN, careful attention was paid to the temperature, antibody concentrations and the quality of the ethanol-NaOH solution. Our results were in agreement with those that we had obtained previously for polyethylene glycol (PEG) sections, suggesting that the present procedure is useful for the detection of FN in Araldite-embedded biological specimens.     

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.     

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.