Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Purification and characterization of tripeptide aminopeptidase from bovine dental follicles

Tripeptide aminopeptidase (EC 3.4.11.4) was purified from bovine dental follicles by a series of chromatographies. Purified enzyme had a specific activity of 59.5 units per mg protein with L-prolyl-glycylglycine as substrate. The pH optimum was 8.0. The purified native enzyme had a Mr of 230,000 and was shown to be a tetramer of subunit Mr of 58,000. The isoelectric point was pH 7.0. The enzyme was inhibited 5-5-dithio-bis (2-nitrobenzoic acid),o-phenanthroline, and bestatin. Substrate specificity studies indicated that the enzyme specifically hydrolyzes the N-terminal amino acid residue from tripeptides only.     

Feulgen nucleal reaction

Summary Electrophoretic means of separation revealed the presence of as many as five reaction products in Schiff-apurinic acid reaction at the maximum. They differed not only in their absorption maxima, but also in their ratios of apurinic acid phosphorus to fuchsin moiety. Some considerations on the reaction mechanism to account for the occurrence of these multiple reaction products have been made. The stoichiometry of Schiff-apurinic acid reaction was studied with respect to the main product responsible for the presentation of reaction color. A reaction product consisting of six or eight atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be formed, provided that the reagent of infinite concentration is used. From theoretical view point, a reaction product consisting of four atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be expected with the reagent of infinite concentration, provided that apurinic acid retains essentially the nucleotide sequence of its parent desoxyribonucleic acid except for some modification of the original purin nucleotide groups to react as aldehyde moieties, and provided that the reaction proceeds at a constant rate irrespective of the concentrations of the reagent.     

Anti-obesity and anti-diabetic actions of a beta 3-adrenoceptor agonist, BRL 26830A, in yellow KK mice.

The anti-obesity and anti-diabetic actions of BRL 26830A, beta 3-adrenoceptor agonist, (2 mg/kg administered intramuscularly daily for 2 weeks) were evaluated in obese diabetic Yellow KK mice and C57B1 control mice. The following parameters were compared in the treated vs. control animals: brown adipose tissue (BAT) thermogenesis, resting metabolic rate (RMR), insulin receptors in adipocytes, and blood glucose and serum insulin levels during a glucose overloading test. BRL 26830A significantly increased BAT thermogenesis and RMR but it decreased the amount of white adipose tissue without affecting food intake. Those actions contributed to the mitigation of obesity in Yellow KK mice. BRL 26830A also increased the concentration of insulin receptors and decreased the levels of serum insulin and blood glucose during the glucose overloading test in Yellow KK mice. In the glucose overloading test performed one hour after BRL 26830A injection, insulin secretion was significantly increased and the blood glucose level was markedly decreased in both groups. These observations suggest that BRL 26830A possesses anti-obesity and anti-diabetic actions and consequently may be useful for treating obesity as well as non-insulin-dependent diabetes mellitus with obesity.     

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Both stimulatory and inhibitory GDP/GTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins.

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.     

Purification of an endothelin receptor from human placenta

We have identified an endothelin (ET) binding protein on the membranes of human placenta and purified it to homogeneity. It is a polypeptide with an apparent Mol. Wt. of 40,000 and is a major protein to be labeled by cross-linking with either 125I-ET-1, -2, or -3. Binding studies with Scatchard analysis indicated the presence of a single class, high-affinity binding site with Kds of 57 pM, 480 pM and 40 nM for 125I-labeled ET-1, ET-2 and ET-3, respectively. These results suggest that the 40K protein is a major ET receptor in placenta and, most likely, can bind differentially to ET-1, ET-2 and ET-3.     

Detection of common flagella antigen in Bacillus cereus by monoclonal antibody

Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined. When the immunochemical method of classification had changed from "agglutination method" to "enzyme-linked immunosorbent assay (ELISA)," the cross-reactivity between each flagella type was increased. These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B. cereus. Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA. Monoclonal antibody provided was specific for B. cereus (H-1 to H-23) and did not show the cross-reactivity with Escherichia coli NIH and Bacillus subtilis.