The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells

Scorpion toxins, the basic miniprotiens of scorpion venom, stimulated the passive uptake of Na⁺ and Ca²⁺ in chick ermbryo heart cells. Half-maximum stimulation was obtained for 20–30 nM Na⁺ and 40–50 nM Ca²⁺. Scorpion toxin-activated Na⁺ and Ca²⁺ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na⁺ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nm) for both Na⁺ and Ca²⁺. Scorpion toxin-stimulated Ca²⁺ uptake was dependent on extracellular Na⁺ concentration and was not inhibited by Ca²⁺ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca²⁺ transport, which is coupled to passive Na⁺ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin — sensitive fast channels.     

The effect of heterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells

The initial process of transfer of extracellular iron to the haem-synthesizing mitochondria of immature erythroid cells is the association of iron-transferrin with the cell membrane. When rat bone marrow cells were incubated in the presence of iron bound to rat transferrin, iron uptake was higher than in the presence of iron bound to heterologous transferrin. The relative activities of the various isolated transferrins towards rat transferrin were found to be approximately 0.3, 0.8, 0.1 and 0.04 for rabbit, human, bovine and fish (tench, Tinca tinca) transferrin, respectively, and 0.7, 0.7 and 0.15 for mouse, guinea pig and calf serum, respectively, as compared with rat serum. Although great difference exist in cellular uptake of iron bound to different species of transferrin, the subcellular distribution of ⁵⁹Fe was quite similar. In all cases about 60% of the radioactivity taken up by the cells could be recovered in the haemin fraction and only about 15% in each the membrane and the non-haem soluble cell fraction. Similar results were obtained with guinea pig bone marrow cells.From the results of the experiments presented it might be concluded that the species of transferrin plays an important role during the initial stages of iron uptake by bone marrow cells, whereas the intracellular iron transfer process is not influenced by the species of transferrin.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria. III. Submitochondrial localization of the iron accumulated

Isolated rat liver mitochondria accumulate iron partly by an energy-dependent and partly by an energy-independent mechanism (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29–40). When the iron-loaded mitochondria were disrupted mechanically and the mitochondrial subfractions isolated by density gradient centrifugation, the iron accumulated by the energy-dependent mechanism was recovered mainly in the soluble matrix and intermembrane space (approx. 50% of the total activity) and the inner membrane (approx. 30%). A negligible contribution to the total iron content of the soluble fraction by intermembrane space was revealed by the preparation of ‘mitoplasts’. On the other hand, most of the energy-independent iron accumulation was confined to the outer and inner membranes (approx. 35% of the total activity in each).     

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.     

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of β-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or α-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [β-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ from 29 to 16 to 10 mM, entry $\text{V}$ being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external β-hydroxybutyrate enhanced efflux slightly. At high external pH, external β-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal β-hydroxybutyrate (βHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled βHB^?/OH^? exchange, βHB^? and OH^? competing for influx, C : OH^? moving faster than C : βHB^?, empty C being immobile. They are also compatible with coupled βHB^?/H⁺ copermeation, empty C moving inward faster than H⁺ : C : βHB^?, H⁺ : C being immobile, and C : βHB^? (without H⁺) being so unstable as not to be formed in significant amounts (relative to C, H⁺ : C, and H⁺ : C : βHB^?).     

Choice of an aqueous polymer two-phase system for cell partition

Partition of human erythrocytes in aqueous two-phase polymer systems produced by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and the influence of the ionic composition on the cells' partition in the systems was studied. It is found that the Ficoll-dextran-40 system is characterized by a number of advantages as compared with the common dextran-polyethylene glycol system or the others systems under study. The main advantage of the system appears to be that it is possible to concentrate the red cells in the top phase or in the bottom phase of the system, depending on the system ionic composition. The influence of the nature and the concentration of salt additives on this two-phase system formation is examined.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

Availability of monovalent and divalent cations within intact chloroplasts for the action of ionophores nigericin and A23187

1. A23187 will uncouple electron transport by broken chloroplasts in a divalent cation dependent manner provided that they have been treated with a low concentration of EDTA.2. A23187 stimulates oxaloacetate-dependent oxygen evolution and inhibits phosphoglycerate reduction by intact chloroplasts isolated in a cation-free medium whereas the full effect of nigericin was dependent on the presence of external K⁺.3. Uncoupling of oxaloacetate reduction by A23187 in intact chloroplasts is inhibited by EDTA and this effect is overcome by excess Mg²⁺.4. The results suggest that divalent and not monovalent cations are available for collapsing the light-induced H⁺ gradient within the intact organelle.     

A specific gastrin receptor site in the rat stomach

A specific receptor for gastrin I has been demonstrated in the rat stomach fundus.Specific binding of ¹²⁵I-labelled gastrin I was localised to particles sedimenting between 250–20 000 × g. Saturation of binding sites occurred with a gastrin concentration of 10^?11 M in an assay system containing 0.6–1.7 mg/ml of homogenate protein. Gastrin binding was shown to be reversible, temperature- and pH-dependent, and susceptible to tryptic digestion. Electron microscopic and enzymatic studies showed the binding fraction to contain predominantly mitochondria. Preincubation of the homogenate with 10^?8 M cholecystokinin or secretin inhibited gastrin binding to a greater extent than an equimolar concentration of pentagastrin. Cimetidine, a histamine receptor antagonist, did not affect binding of gastrin to the receptor.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.