Selective modification of myosin SH1 with 1,2,4-trinitrobenzene. VIII. Thiols of myosin

Myosin has 2 mol of the most reactive thiol, named SH1. 1,2,4-Trinitrobenzene (TNB), a novel dinitrophenyl(DNP)ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the lambda max of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8 M urea. This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.     

Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.     

Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides

A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo.     

S100a0 (αα) Protein Is Present in Neurons of the Central and Peripheral Nervous System

The cellular distribution of S100 subunits in human brain and peripheral nerves was studied by means of an immunohistochemical technique using antibodies specific to the alpha subunit or the beta subunit of S100 protein. The results indicate that the distribution of the alpha subunit and the beta subunit is different among cell types in the nervous tissue, and that neurons in the brain and peripheral nerves contain only the alpha subunit, or S100a0 protein. The subunit distribution also appears to be different at an intracellular level, where the immunoreaction products for the alpha subunit show granular arrangement whereas those for the beta subunit are found diffusely in the cytoplasm.     

Chemical modification of lipase with ferromagnetic modifier — A ferromagnetic-modified lipase

Summary A ferromagnetic modifier was prepared by reacting ferrous(Fe²⁺)- and ferric(Fe³⁺)-ions with polyethylene glycol having two carboxyl groups (MW:2000) at pH 8.0–8.5. Lipase fromPseudomonas fragi 22–39B was coupled with the modifier using water-soluble carbodiimide. The modified lipase, which was dispersed into buffered solutions in the size range of 30–70 nm, exerted the hydrolytic activity of 8.0 U/mg. In a magnetic field of 250 Oe, the ferromagnetic-modified lipase was readily recovered from the colloidal solution.     

Dietary selenium status and plasma thyroid hormones in chicks

Experiments were conducted to study the effect of marginal levels of selenium and vitamin E on plasma thyroid hormones of meattype chicks. Plasma thyroxine (T₄) was significantly increased when a semipurified diet was supplemented with either selenium or vitamin E. Triiodothyronine (T₃) was also significantly increased by vitamin E and in one experiment with selenium supplementation. No significant increase in these hormones was observed in birds fed a corn-soybean-meal diet supplemented with these nutrients. Plasma corticosterone level was reduced and weight of the bursa of Fabricius increased by selenium or vitamin E supplementation. These nutrients may be necessary for providing the optimum thyroid conditions for activity of thyroid peroxidase.     

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)