Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

The Effect of Electrotonus on the Olfactory Epithelium

The effect of electrotonus on the slow potential of the olfactory epithelium of the frog was studied. The "on"-slow potential induced by a general odor like amyl acetate increased its magnitude in accordance with increase of anodal current, while it decreased its magnitude with increase of cathodal current. Similar relations were also found in the case of the vapors of organic solvents like ethyl ether of low concentrations. Conversely, the on-slow potential induced by the vapors of organic solvents of high concentration decreased its magnitude in accordance with the increase of anodal current, while it increased its magnitude with the increase of cathodal current. The "off"-slow potential induced by the vapors of organic solvents of high concentration showed a potential change under the action of electrotonic currents which is similar to the change of the on-slow potential induced by general odors. It was concluded that there are two receptive processes in the olfactory cell. One is an ordinary excitatory process which produces an electronegative slow potential in response to general odors. The other is a process of a different kind which is activated only by the vapor of an organic solvent of high concentration and which shows an entirely opposite reaction from that generally found in excitable tissues when an electrotonic current is applied.     

Identification of glycosphingolipid-glycoprotein hybrids in gastric epithelium

A glycolipid fraction, obtained from dog gastric epithelium by detergent solubilization, was found to contain the glycoconjugates with features common to both glycosphingolipids and glycoproteins. Three such compounds have been purified to homogeneity and their composition and structural characteristics determined. The purified glycosphingolipid-glycoprotein hybrids had a molecular weight in the range of 14,000 and contained 88.2 - 91.1% carbohydrate, 3.1 - 4.5% protein, 2.0 - 2.4% sphingosine, and 1.1 - 1.9% fatty acids. The oxidation of the olefinic bond of sphingosine followed by beta-elimination reaction led to the release from each compound of glucose and mannose containing oligosaccharides. Deglycosylation with trifluoromethanesulfonic acid exposed the core regions of the hybrid molecules which were found to consist of sphingosine, glucose, N-acetylglucosamine and amino acids. The data suggest that glycosphingolipid-glycoprotein hybrid molecules are held together by amide linkages between the sphingosine and aspartic or glutamic acid.     

Morphine-like analgesic actions of enkephalin-like peptides containing the Tyr-Arg unit

The analgesic actions of some synthetically prepared peptides having the Tyr-D-Arg unit at the N terminal portion of met- and leu-enkephalin were measured by the intra-cisternal injection method in mice. Among them, Tyr-D-Arg-Gly-Phe (DR-4) induced the most potent naloxone-reversible analgesia and was also effective by s.c. injection. DR-4 showed the good affinity to mu-receptor, and the resistance to the enzymatic degradation.     

Immunological detection of propolypeptide of von Willebrand factor on platelet surface

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.     

Primary structure of two linker chains of the extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

Two types of linker subunits (linkers 1 and 2) of the extracellular hemoglobin of Tylorrhynchus heterochaetus have been isolated as disulfide-linked homodimers by C18 reverse-phase chromatography. These subunits constituted 6 and 13%, respectively, of total protein area on the chromatogram. The complete amino acid sequences of linkers 1 and 2 were determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, pepsin, and endoproteinase Asp-N. The linker 1 consisted of 253 amino acid residues (the calculated molecular mass, 28,200 Da), while the linker 2 consisted of 236 residues (26,316 Da). The two chains showed 27% sequence identity. The amino acid sequences of Tylorrhynchus linkers 1 and 2 also showed 23-27% homology with the recently determined sequence of a linker chain of Lamellibrachia hemoglobin (Suzuki, T., Takagi, T., and Ohta, S. (1990) J. Biol. Chem. 265, 1551-1555). In the three linker chains, half-cystine residues were highly conserved; 8 out of 13 residues are identical, suggesting that such residues would contribute to the formation of intrachain disulfide bonds essential for the protein folding of the linker polypeptides. Based on the exact molecular masses of the linker and the heme-containing subunits, the molar ratios estimated for the subunits and the minimum molecular weights per 1 mol of heme, a model is proposed for the subunit structure of the Tylorrhynchus hemoglobin, consisting of 216 polypeptide chains, 192 heme-containing chains, and 24 linker chains.     

Nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions.

The nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions has been determined. The homologies of the 5.8S rRNA sequence with those of Saccharomyces, Chlamydomonas and Xenopus were 56%, 50% and 52%, respectively. In spite of these relatively low homologies, its possible secondary structure was very similar to those of other species.     

Study of oxytocin receptor: II. Gestational changes in oxytocin activity in the human myometrium

This study was designed to investigate the oxytocin (OT) specific binding receptors in 20,000 x g pellets of nonpregnant, first trimester and term human myometria. The receptor analysis was done using the lower uterine segment at term and the lower portion of the anterior uterine body in nonpregnant and first trimester subjects, and no difference was found in the myometrial receptor concentrations in the various uteri. The mean +/- S.D. values of the receptor dissociation constants were 3.33 +/- 0.50, 2.71 +/- 1.03 and 1.87 +/- 0.30 nM and the number of binding sites was 0.30 +/- 0.10, 0.50 +/- 0.10 and 1.50 +/- 0.50 pmol/mg protein at each stage studied, indicating that the gestational increase of uterine sensitivity to OT is due to the increase in myometrial OT binding sites as well as its binding affinity. Further, myometrial OT binding before and after the onset of labor was studied and a marked decrease in total myometrial OT binding was noticed; 35.6 +/- 13.0% before and 20.2 +/- 5.0% after. This decrease was thought to be due to the decrease in the number of binding sites from 1.50 +/- 0.50 to 0.74 +/- 0.21 pmol/mg protein after the onset of labor (p less than 0.01). No changes were found in the dissociation constants. Thus it seems that OT and its receptor coupling triggers labor or is involved in the early steps of labor.