Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor,peripheral ν-polypeptide (Mr 43,000)

NaIO₄ oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (α, β, γ and δ) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ν-peptide (M_r 43,000), pH and temperature. Very low NaIO₄ concentrations (50 μM) suffice to prevent the penetration of the ν-peptide into NaDodSO₄ polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (α, M_r 40,000) into a doublet. In contrast, NaIO₄ treatment of the detergent-solubilized receptor aggregated the α-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO₄-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (～2 mM NaIO₄).Neuraminidase digestion affected exclusively the γ- and δ-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. β-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO₄ nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-α-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and β-glucosidase treatments. The latter enzymatic digestion reduced the α-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.     

Developmental studies on creatine kinase. Isoenzyme in rat gastrointestinal tract

Creatine kinase activity and its isoenzymatic profile in rat intestinal mucose during normal development have been studied. Creatine kinase enzymatic activity increased stepwise during fetal development and the first week of life. An isoenzymatic pattern of exclusively CK-BB types occurred in all segments of the digestive tract during the early fetal stage. The isoenzyme profile of creatine kinase in the esophagic tissue with advancing maturation of the fetus shifted in the same way as in adults, with preferential concentration of CK-MM. However, CK-BB continued to be the main isoenzyme in the rest of the digestive tract. Our results show that rats are particularly suitable for experimental studies of intestinal creatine kinase isoenzymes.     

Influence of blood proteins in the in vitro adhesion of Staphylococcus epidermidis to teflon, polycarbonate, polyethylene and bovine pericardium.

The influence of human plasma proteins (fibrinogen, albumin and fibronectin) on the adherence of Staphylococcus epidermis to teflon, polyethylene, polycarbonate and bovine pericardium was studied in an in vitro quantitative assay by scintillation counting. Bacterial adhesion was generally reduced by the presence of protein during the adherence assay except in the case of bovine pericardium, in which adherence remained almost unaffected. The effect of these plasma proteins on bacterial surface properties resulted in strong increases of surface charge as measured by ion-exchange chromatography and with no effect on hydrophobicity, estimated as contact angles. Adherence was not found to be correlated with these two properties, suggesting that bacteria-surface interactions must not be simplified to the influence of interfacial forces.     

Analysis of the molecular mass heterogeneity of the transferrin receptor in Neisseria meningitidis and commensal Neisseria

Peroxidase-conjugated transferrin was used to detect transferrin receptors both in intact outer membrane vesicles (OMVs) from Neisseria species in a dot blot assay, and in SDS-PAGE-separated OMV proteins after transferring to nitrocellulose membranes. All N. meningitidis strains produced transferrin receptors after culturing in either iron sufficiency or iron restriction although expression was higher in the latter case, whereas only six N. lactamica and two N. sicca (among 20 commensal species) were able to bind transferrin. Molecular mass (MM) of the receptors were mainly between 78 kDa and 85 kDa (87.5% of strains), 12.5% had receptors with MM close to 70 kDa, and 5% showed receptors with MM over 85 kDa. Our results confirm the molecular mass heterogeneity of the transferrin receptors in N. meningitidis, completely disagree with the 'universal' 98 kDa receptor proposed by some authors, and show a low expression of the receptor in commensal Neisseria.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of a K99-antigen associated adhesin in Escherichia coli (637 strain)

The K99-antigen associated adhesin in Escherichia coli (637 Strain) has been purified to homogeneity by using conventional chromatographic procedures. Sodium deoxycholate was used in the precipitation steps to avoid hydrophobic interactions between the fimbriae and other membrane-associated components. Homogeneity of the purified adhesin was assessed by electrophoresis, isoelectrofocusing, analytical gel filtration and immunoprecipitation against K99 specific antiserum, being homogeneous in all cases. The purified adhesin is composed of protein sub-units with a molecular weight of 18,900 +/- 950 daltons. No sugars were detected in the molecule. The molecular weight of the adhesin was higher than 2 X 10(6) daltons, and its isoelectric point was estimated to be about 9.45.     

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Tr?uble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.     

Quantitative and qualitative finger dermatoglyphics in the Basque Valley of Urola, Spain

Digital dermatoglyphics of an indigenous sample of 87 males and 101 females from the Urola Valley in the Spanish Basque Country are compared with those from the nine other Basque valleys previously analyzed. In both sexes of the Urola Valley, there is a very high frequency of radial loops; the mean found in this valley extends the range of variation for South European-Mediterranean populations, and it is in the superior limit of all Caucasian populations. There are bimanual and sex differences in the frequencies of whorls and loops. Contrary to what occurs normally in populations, in the Urola Valley the frequency of whorls is higher in females and the quantitative value of digital patterns is lower in males. The results of this study show the existence of heterogeneity among valleys for digital trait frequencies and for finger ridge count, and this heterogeneity is more marked in females.     

Method for Lyophilizing Brain Proteolipid Preparation That Increases Subsequent Solubilization by Detergent

Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.