A theoretical study of the dielectric constant of protein

The dielectric properties of a protein molecule were investigated by calculating a 'local dielectric constant' with the aid of normal mode analysis. This local dielectric constant was calculated from the electronic polarization of atoms and the orientational polarization of local dipoles. The former was obtained from atomic polarizations of the whole atoms in a protein molecule. The latter was determined from the fluctuation-dissipation theorem. The degree of dipole fluctuation was calculated from the positional fluctuation of each atom obtained by the normal mode analysis. Assuming a minimum volume for a continuum model, the resulting local dielectric constants ranged from 1 to 20 inside the protein.     

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).     

Model study on the strain and stress distributions in the vicinity of an arterial stenosis

By the evaluation of the strain and stress distributions in the vicinity of a stenosis, it is suggested that the bending moment generated by the axial force acting on a stenosis is one of the causes of the post-stenotic dilatation. The conditions which enhance this bending moment are investigated and it is expected that the present mechanism is specially effective for the artery where the ratio of wall thickness to radius is very small. Lastly, the concrete numerical value of this bending moment is evaluated and it is shown that the bending moment generated by this mechanism is large enough to cause the post-stenotic dilatation.     

Complete structural analysis of globoseries glycolipids by two-dimensional nuclear magnetic resonance

Combined two-dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two-dimensional chemical-shift-correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two-dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two-dimensional J-relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis

Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3-4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly-Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+-ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase.     

Mutations upstream of the ribosome-binding site affect translational efficiency

The DNA coding for the major outer membrane lipoprotein of Escherichia coli has been fused to the coding region of the beta-galactosidase gene to measure the effect of various mutations on the efficiency of translation initiation. The various mutants were made by either inserting or deleting a small number of nucleotides into or from a region just upstream of the ribosome-binding site. These small mutations dramatically affect translation initiation as measured by the production of beta-galactosidase. We postulate that these mutations affect translation initiation by altering the secondary structure of the messenger RNA. In one case, we predict that a stem and loop just upstream of the Shine-Dalgarno sequence sterically hinders the binding of the ribosome to the mRNA.