Interaction between gramicidin-A and bacteriorhodopsin in reconstituted purple membrane

The addition of gramicidin-A to reconstituted purple membrane, significantly inhibits light-induced proton movement. Kinetic analyses indicate that the treatment decreases the initial proton pumping rate (R_o), alters the interdependence (m) between the pumping process and its associated H⁺ leak path (k_L-k_D), but has no detectable effect on the proton permeability associated with phospholipid bilayers in the dark (k_D). These results suggest that gramicidin-A, under the experimental conditions, interacts directly with bacteriorhodopsin in the membrane. This suggestion is supported by the findings that both the resonance Raman and circular dichroism spectra of bacteriorhodopsin are affected by the antibiotic.     

Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture

The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-³H]cytochalasin B and transport of $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-[}{}^{14}\text{C]glucose}$ into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a K_D of 2.86 · 10^?7 M, while the low-affinity site had a K_D of 1.13 · 10^?5M. Sugar transport was monitored by $\text{3-O-}\text{methyl}\text{-}\text{D}\text{-}\text{glucose}$ uptake and it was found that cytochalasin B (10^?5M) drastically inhibited transport. However, D-glucose (10^?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Treatment of mobile phase particulate matter in low-angle quasi-elastic light scattering

The method of quasi-elastic laser light scattering (QLS), particularly at low forward scattering angles, has been complicated by the transient presence of Mie or large Rayleigh scattering particles which contaminate the scattering volume. These large contaminating particles have substantial effects on photon correlation spectroscopy because the presence of these larger scatterers tends to decrease the value of the apparent diffusion coefficient of the particle of interest. A method is presented which yields more accurate diffusion constants by autocorrelation of selected photon count periods representative of minimal Mie or large Rayleigh particle contamination. This method was applied to the determination of the apparent diffusion constant for four proteins—ovalbumin, chymotrypsinogen-A, bovine serum albumin, and ribonuclease-A.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Rapid isolation of pancreatic islets from collagenase digested pancreas by sedimentation through Percol at unit gravity.

A Percoll solution with a density of 1.045 g/ml was used to separate pancreatic islets and exocrine tissue from collagenase-digested human and $\text{ob/ob}$ mouse pancreases by sedimentation at unit gravity. Most exocrine tissue from the mouse was found to range in density from 1.015 to 1.045 g/ml whereas the denser islets lay in a narrower range of 1.065–1.070 g/ml. Up to 400 islets were obtained from each mouse pancreas and 140 islets from 4 g of human pancreas; the isolated islets being essentially free from contamination with exocrine tissue. Glucose-stimulated insulin release was the same whether the mouse islets were isolated with or without Percoll. The simplicity of the method makes it suitable for large-scale islet isolation, a feature of potential importance for the treatment of diabetes by islet transplantation.     

Reconstitution of succinate-Q reductase.

Reconstitution of succinate-Q reductase is achieved by admixing soluble succinate dehydrogenase (SDH) and ubiquinone-protein-S (QP-S), a new protein isolated from the soluble cytochrome $\text{b-c}{}_1$ complex. The reconstituted reductase catalyzes reduction of Q by succinate. The reaction is fully sensitive to thenoyltrifluoroacetone. The reconstituted reductase (same as succinate-cytochrome $\text{c}$ reductase or submitochondrial particles) does not show “low concentration ferricyanide reductase activity” as soluble dehydrogenase does. In other words, this enzymic site on SDH is occupied by QP-S. When an artificial dye, such as phenazine methosulfate or Wurster's Blue, is used as electron acceptor the rate of oxidation of succinate by SDH is not significantly changed regardless of whether the dehydrogenase is in the free or in the reconstituted succinate-Q reductase forms.     

Structural differences between ribosomes of various eukaryotes: stability, density, mass, size and structure in solution of cytoplasmic ribosomes from Tetrahumena, Artemia and Euglena

Cytoplasmic ribosomal particles were isolated from the ciliated protozoa Tetrahymena pyriformis, the brine shrimp Artemia, and the flagellated alga Euglena gracilis; their stability, density, mass, size and structure in solution have been compared in identical conditions. The small and in particular the large subunits of Euglena ribosomes were less stable than the corresponding particles of the other eukaryotes. Equilibrium centrifugation of the ribosomes of the various species in CsCl density gradients revealed no difference in either their homogeneity or their buoyant density; so no differences were detected in this way in the ratio of their RNA to protein content or in their partial specific volume. Using analytical boundary sedimentation, the sedimentation coefficients s_20,w⁰ of the whole ribosomes were measured to be 81, 81 and 86 S, respectively. Photon correlation spectroscopy of laser light scattered by the whole ribosomes has yielded values for their diffusion coeffcient D_20,w⁰) of 1.51, 1.42 and 1.29 × 10⁻⁷ cm² s⁻¹, respectively; this corresponds to values for their hydrodynamic radius of 142, 151 and 166 Å, respectively. The data indicate also that the solvation of the ribosomes increases with their size and mass.So evidence has been obtained for differences in the size of various eukaryotic ribosomes, and even in the degree of compactness of their structure in solution.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Alkaline opening of imidazole ring of 7-methylguanosine. 2. Further studies on reaction mechanisms and products

High performance liquid chromatography (HPLC) was used to follow the kinetics of the alkaline induced opening of the imidazole ring of 7-methylguanosine (7-meGuo). The kinetics show an initial rapid formation of a major transient intermediate and some minor products that were chromato-graphically separable into seven peaks. This phase of the reaction is followed by the formation of a dominant pyrimidine derivative whose liquid chromatography retention time in a 6% methanol, 0.01 M NH₄H₂PO₄ (pH 5.1) solvent is 6 min; during the rest of the reaction time this dominant species was progressively converted to a co-dominant species that has a 4.5-min column retention. Mass spectroscopy confirmed the existence of two species of ring opened 7-methylguanine (7-meGua), one formylated and another deformylated. Schiff's reaction demonstrated that the species in the second HPLC peak is the formylated one. The ring opened 7-methylguanine (rom⁷Gua) released by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species. These results demonstrate that the enzyme excises formylated rom⁷Gua from DNA Analysis of rom⁷Guo by NMR showed that there are two signals assignable to methyl protons and two to formyl protons. These chemical shifts were interpreted as being due to the opening of the imidazole ring at two sites and to the formation of formylated and deformylated rom⁷Gua.     

Kinetics of hydrogen-deuterium exchange in ATPase from a thermophilic bacterium PS3.

The kinetics of the hydrogen-deuterium exchange reaction in a stable ATPase (TF₁) from a thermophilic bacterium PS3 was followed by infrared absorption measurements. The rates of the hydrogen-deuterium exchange reactions decreased in following order; free form, TF₁·ADP, TF₁·ATP and TF₁·AMP-P(NH)P. TF₁ does not dissociate into subunits even in the absence of nucleotides, thus differences in exchange likely reflect differences in conformations of subunits. These results indicate that the structure is most restricted when ATP or AMP-P(NH)P is bound to the enzyme.     

On the activity of ribulosediphosphate carboxylase with CO2 and O2 from leaf extracts of Zea mays

Ribulosediphosphate carboxylase, partially purified from corn leaves, demonstrates a low K_m(CO₂) of 19 μM if stabilized with ribose-5-phosphate during extraction. It also exhibits a ribulosediphosphate dependent uptake of oxygen, similar to that observed with spinach carboxylase. The low K_m(CO₂) is similar to the apparent K_m(CO₂) for photosynthesis by intact corn tissue and requires reconsideration of the hypothesis that CO₂ is concentrated in the bundle sheath cell by the C₄ pathway during photosynthesis.     

Protective effect of EGTA on rat gastric membranes enriched in (H+-K+)-ATPase

Rat gastric membranes enriched in (H⁺-K⁺)-ATPase, when prepared in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid, showed the ability to accumulate H⁺ ions upon addition of ATP, KCl, and valinomycin. The membranes were largely impermeable to K⁺ and Cl^?. In contrast, the rat membranes prepared without the Ca²⁺ chelator lost the ability to develop a pH gradient because of the membrane leakiness to H⁺. A majority of these membrane vesicles became also permeable to K⁺. We suggest that the calcium chelator preserved the gastric membrane permeability barrier during isolation by inhibiting various Ca²⁺-dependent phospholipases in rat gastric mucosa.     

Developmental changes in the form of the Arrhenius plots of rat liver plasma membrane adenylate cyclase and 5'-nucleotidase activities

Human α-1-proteinase inhibitor is inactivated by human myeloperoxidase in the presence of hydrogen peroxide and chloride ion. Several antiarthritic drugs and related compounds, including many containing gold, were tested as inhibitors of the myeloperoxidase system. Of the twenty-six compounds used, twenty-two inhibited. The most important feature of these was the presence of a sulfhydryl group. The most effective compounds also were the most hydrophobic. The presence of gold, on the other hand, made little difference to the amount of inhibition. These drugs appear to have many effects, and their inhibition of the myeloperoxidase system suggests that this could be one of them.