Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding

A regulatory role for cytoplasmically derived proteins in chloroplast translation in organello was examined by analyzing protein synthesis in plastids isolated from cells of Euglena gracilis which had been treated with cycloheximide (CHI). Incorporation of [35S]methionine by chloroplasts from CHI-inhibited Euglena was reduced approximately 40 and 90% by exposure of the cells to the antibiotic for 2 and 4 h, respectively. The chloroplast translation products were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of polypeptides in the soluble compartment of the plastid was substantially diminished by as little as 15 min of CHI pretreatment. No qualitative alterations of the polypeptide pattern were detected. Qualitative changes were seen in the thylakoid fraction, however. Comparison of the stainable polypeptides and fluorographs of thylakoid membranes from CHI-treated cells with those of controls showed several instances in which the more slowly migrating member of a doublet accumulated with a concomitant depletion of a more rapidly migrating component. A pair of polypeptides at 28 and 30 kDa, which we believe are the Euglena homologs of the photogene product and its precursor, respectively, are representative of this phenomenon. Additionally, thylakoids from cells pretreated with CHI sometimes synthesized novel polypeptides larger than 65 kDa. Finally, when intact chloroplasts from CHI-inhibited Euglena were incubated with a postchloroplast supernatant from normal cells, there was a partial reversion of the anomalies seen in the fluorographs. These data are interpreted to indicate the cytoplasmic origin of one or more proteins whose function is to process chloroplast translation products.     

Stimulatory effect of calcium on photoactivation of the water oxidation system in dark-grown spruce chloroplasts

Treatment of platelets or red cells with small amounts of phospholipase C from Clostridium welchii enables both cells, prior to the onset of lysis, to stimulate prothrombin conversion by coagulation factor Xa and Va in the presence of calcium. Phospholipase C treatment of both cells also exposes significant amounts of phosphatidylserine at the outer surface. The level of phosphatidic acid formed from diglycerides produced by phospholipase C action, is similar to that formed in activated platelets upon triggering the phosphatidylinositol cycle. A possible involvement of this cycle to activate platelets to become more procoagulant is discussed.     

3H]Captopril binding to membrane associated angiotensin converting enzyme

[3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a KD of 2.4 nM. [3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [3H]captopril binds selectively to angiotensin converting enzyme. [3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.     

Cell wall receptor for yeast killer toxin: involvement of (1 leads to 6)-beta-D-glucan

The linear (1 --> 6)-beta-d-glucans pustulan and luteose were effective competitive inhibitors of killer toxin action. Affinity chromatography of killer toxin on a pustulan-Sepharose column showed that toxin bound directly to a (1 --> 6)-beta-linked polysaccharide. Other polysaccharides found in yeast cell walls, including (1 --> 3)-beta-d-glucan, mannan, chitin, and glycogen, were not effective as inhibitors of toxin. Fractionation of yeast cell walls was attempted to identify the toxin receptor in sensitive Saccharomyces cerevisiae. The receptor activity was retained among the insoluble glucans in alkali-washed cells; yeast mannan and alkali-soluble glucan had little receptor activity. A minor fraction of receptor activity was removed from alkali-washed cells by hot acetic acid extraction, a procedure which solubilized some (1 --> 6)-beta-d-glucan and glycogen. The major fraction (>70%) of receptor activity remained with the acid-insoluble (1 --> 6)-beta-and (1 --> 3)-beta-glucans. Zymolyase, an endo-(1 --> 3)-beta-d-glucanase, solubilized a substantial fraction of the receptor activity in the acid-insoluble glucans. The receptor activity in yeast cell walls was periodate and (1 --> 6)-beta-d-glucanase sensitive, but was resistant to (1 --> 3)-beta-d-glucanase and alpha-amylase. The acid-soluble glucan fractions of a sensitive strain and a krel-l receptor-defective toxin-resistant mutant were examined. The krel-l strain had a reduced amount (ca. 50%) of (1 --> 6)-beta-d-glucan compared with the sensitive parent strain. A sensitive revertant of the krel-l strain regained the parental level of glucan. These results implicate (1 --> 6)-beta-d-glucan as a component of the yeast cell wall receptor for killer toxin.     

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an α₂β₂ structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M⁻¹ cm⁻¹, respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the α and β subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70°C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65°C, and the Q₁₀ values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.     

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.     

On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.