Yoshiro Ogoma

Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r.*

The interactions of the S100 protein (S100) with metal cations such as Ca2+, Mg2+, Zn2+ and K+ were studied by the metal n.m.r. spectroscopy. The line widths of 43Ca, 25Mg, 67Zn and 39K n.m.r. markedly increased by adding all S100s. A broad 43Ca n.m.r. band of Ca(2+)-S100a solution was not affected by Zn2+ and K+, while it was greatly decreased by adding Mg2+. The 43Ca n.m.r. spectra of Ca(2+)-S100a0 and -S100b solutions consisted of two slow-exchangeable signals which corresponded to Ca2+ bound to two environmentally different sites of the S100a0. These two 43Ca n.m.r. signals were not affected by Zn2+ and K+. The line width of broad 25Mg n.m.r. band of the Mg(2+)-S100 solution greatly decreased by adding Ca2+, while it did not change by adding Zn2+ and K+. Further, the addition of Ca2+, Mg2+ and K+ did not affect the line width of the 67Zn n.m.r. of the Zn(2+)-S100 solutions. These findings suggest that: (1) Mg2+ binds to all S100s, and at least one of the Mg2+ binding sites of S100 molecule is the same as the Ca2+ binding site; (2) Zn2+ binds to S100s, although the binding site(s) is/are different from Ca(2+)- or Mg(2+)-binding site(s), and the environment of Zn2+ nuclei will not change even though Ca2+ binds to S100s.

Selective purification of microtubule-associated proteins 1 and 2 from rat brain using poly(l-aspartic acid)☆

A rapid and selective purification procedure for microtubule-associated protein (MAP) 1 and MAP 2 has been established. This procedure is based upon the fact that poly(L-aspartic acid) (PLAA) can specifically remove MAP 1 from microtubules polymerized by taxol (Nakamura et al., 1989, J. Biochem. 106, 93-97). MAP 1 released by PLAA was further purified by column chromatography on phosphocellulose and Bio-Gel A-15m. The purified MAP 1 contained MAPs 1A and 1 B. From microtubules devoid of MAP 1, MAP 2, consisting of MAPs 2A and 2B, could also be isolated by exposure to high ionic strength solutions in the presence of taxol without heat treatment. Both MAPs 1 and 2 cosedimented with microtubules consisting of purified tubulin.

The combined effects of transcutaneous electrical nerve stimulation (TENS) and stretching on muscle hardness and pressure pain threshold

[Purpose] This study aimed to clarify the immediate effects of a combined transcutaneous electrical nerve stimulation and stretching protocol. [Subjects] Fifteen healthy young males volunteered to participate in this study. The inclusion criterion was a straight leg raising range of motion of less than 70 degrees. [Methods] Subjects performed two protocols: 1) stretching (S group) of the medial hamstrings, and 2) tanscutaneous electrical nerve stimulation (100 Hz) with stretching (TS group). The TS group included a 20-minute electrical stimulation period followed by 10 minutes of stretching. The S group performed 10 minutes of stretching. Muscle hardness, pressure pain threshold, and straight leg raising range of motion were analyzed to evaluate the effects. The data were collected before transcutaneous electrical nerve stimulation (T1), before stretching (T2), immediately after stretching (T3), and 10 minutes after stretching (T4). [Results] Combined transcutaneous electrical nerve stimulation and stretching had significantly beneficial effects on muscle hardness, pressure pain threshold, and straight leg raising range of motion at T2, T3, and T4 compared with T1. [Conclusion] These results support the belief that transcutaneous electrical nerve stimulation combined with stretching is effective in reducing pain and decreasing muscle hardness, thus increasing range of motion.

Interaction of trifluoperazine with S100 protein: a 19F NMR study

Abstract 19 F NMR spectra were measured to investigate the interaction of trifluoperazine (TFP) with porcine brain S100 protein (S100) under various conditions. It was found that TFP binds to S100 irrespective of Ca 2+ . However, in the presence of Ca 2+ the apparent affinity of TFP to protein ( K d = 20 μM) was greater than that in its absence ( K d = 85 μM). Zn 2+ also enhanced the binding of TFP to S100. The ratio of TFP bound to S100 was estimated to be nearly unity in the presence of Ca 2+ . It was also found that KCl only markedly affected the interaction of TFP with S100 in the presence of Ca 2+ . The 19 F NMR chemical shift of the TFP-S100 solution changed much depending upon the pH of the solution in the presence of Ca 2+ , while no remarkable pH dependence of the 19 F NMR chemical shift was observed for the TFP-S100 solution in the absence of Ca 2+ . These pH effects are in contrast wih those observed for the TFP-calmodulin solution.

Conformation of polyamino acids containing fluorine

Abstract The conformation of poly(γ-4-fluoro-benzyl- l -glutamate) (F-PBLG) and poly(γ-4-trifluoromethyl-benzyl- l -glutamate) (CF 3 -PBLG) was investigated. Circular dichroism (c.d.), infra-red (i.r.) spectra and X-ray analysis indicated that the secondary structure of F-PBLG and CF 3 -PBLG was α-helical both in the solid state and in chloroform solution, similar to PBLG, while it was random coil in trifluoroacetic acid (TFA) and dichloroacetic acid (DCA). In the chloroform-TFA solvent system, these polypeptides changed their conformation from helix to coil; the minimum TFA concentrations required for helix-coil transition were 10–12% in CF 3 -PBLG, 14% in F-PBLG and 12–13% in PBLG. The results suggest that CF 3 groups have a pronounced effect on the stability of α-helical structure of these polypeptides. The helix-coil transition of the polypeptides was accompanied by a perturbation of 19 F nuclear magnetic resonance chemical shift.

19F-n.m.r. study of trifluoperazine-S100 protein interaction: effects of Ca2+ and Zn2+

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.