Specific binding of the calcium-dependent regulator protein to brain membranes from the guinea pig

The interaction of a calcium-dependent regulator protein (CDR) of brain adenylate cyclase (EC 4.6.1.1) with synaptic membranes from guinea pig brain was examined using ¹²⁵I-CDR as a tracer molecule. ¹²⁵I-CDR binding was reversible, saturable, and temperature sensitive. The same Ca²⁺ and Mg²⁺ dependence was observed for ¹²⁵I-CDR binding and for brain adenylate cyclase activation by CDR.     

Trypsin and calcium ions elicit changes of the membrane potential in pig blood platelets.

The addition of trypsin or thrombin or of Ca²⁺ ions to pig blood platelets was followed by a K⁺- dependent change of the membrane potential similar to that produced by the ionophore valinomycin. The effect of trypsin and of Ca²⁺, but not of valinomycin, was prevented by La³⁺ and by EGTA. It is proposed that upon the modification of the platelet surface by trypsin (and by thrombin under physiological conditions) membrane Ca²⁺ move from the external to the internal side of the platelet surface membrane and open the gates of K⁺ - specific channels.     

Sealed lysimeters for the direct estimation of dinitrogen fixation by grain legume crops

Abstract A new technique has been devised for the direct estimation of the contribution of N₂-fixation to the total nitrogen of a legume crop. Sealed lysimeters and ancillary equipment are described by which it is possible to enclose in a gas-tight system the roots of some of the plants within the crop, together with their associated core of soil. The normal soil atmosphere can then be replaced by one containing ¹⁵N₂, thus allowing, from the ¹⁵N content of the resulting plants direct calculation of the N₂-fixation. Regular monitoring is necessary to ensure that soil O₂, CO₂ and moisture contents are maintained at normal field levels. The results indicate that the technique is capable of achieving its objectives and, provided the seedlings establish well initially, the resultant plants fully match the field average at final harvest. It has been possible to maintain the labelling of the soil atmosphere sufficiently constant to ensure that reliable and highly reproducible estimates of N₂-fixation are obtained. Using Pisum sativum cv. Meteor at densities of 160 plants m^?2, fixation accounted for about 90% of the total nitrogen uptake. The limitations and merits of the method are compared with those of the ¹⁵N-fertilizer dilution method.     

Membrane reconstitution in chl-r mutants of Escherichia coli K 12.: VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles

Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5 % polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg²⁺ dependent, insensitive to inhibition by $\text{N,N′-}\text{dicyclohexylcarbodiimide}$ and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg²⁺ are able to incorporate the purified ATPase only in the presence of 2–6 mM Mg²⁺. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increases in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.     

Na+, K+-ATPase: evidence for the binding of ATP to the phosphoenzyme

Highly purified Na⁺, K⁺-ATPase of the dog kidney was reacted with Mg²⁺+³²Pi or Mg²⁺+³²Pi + ouabain. ³²P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K_0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na⁺. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested.     

Subunits of the calcium ion-pump system of sarcoplasmic reticulum

Sarcoplasmic reticulum membranes with high content of Ca²⁺-ATPase (80% of total protein) were dissolved in a non ionic medium and were submitted to isoelectric focusing in polyacrylamide gels. The membrane protein was resolved into six main bands whose isoelectric points range from 6 to 5. The mol. wt. of these peptides is about 100 000 as estimated by second dimension electrophoresis in sodium dodecyl sulfate-polyacrylamide system. The electrophoretic behaviour of the purified ATPase enzyme is similar to that of crude membranes.     

1-Butanol extracted proteolipid. Proton conducting properties

The 1-butanol extracted proteolipid from mitochondria was incorporated to liposomes. This proteolipid mediates the H⁺ transfer across the lipid bilayer in response to a negative charge produced by valinomycin and KCl. The process is sensitive to DCCD, but not to oligomycin. The flux of H⁺ depends on the concentration of proteolipid and the inhibition of this flux depends on the concentration of DCCD.     

Studies on the reactions of ruthenium(II) substrates with tridentate (N,N,O) azo ligands

Reactions of 1-{[2-(arylazo)phenyl]iminomethyl}-2-phenol, HL_sal, 1, [where H represents the dissociable protons upon complexation and aryl groups of HL_sal are phenyl for HL¹_sal, p-methylphenyl for HL²_sal, and p-chlorophenyl for HL³_sal], ligands with Ru(H)(CO)(Cl)(PPh₃)₃ afforded complexes of composition [(L_sal)Ru(CO)(Cl)(PPh₃)] and (L_sal)₂Ru where the N,N,O donor tridentate (L_sal)⁻ ligands coordinated the metal centre facially and meridionally, respectively. Stepwise formation of [(L_sal)₂Ru] has been ascertained. Reaction of 1-{[2-(arylazo)phenyl]iminomethyl}-2-napthol, HL_nap, 2, [where H represents the dissociable protons upon complexation and aryl groups of HL_nap are phenyl for HL¹_nap, p-methylphenyl for HL²_nap, and p-chlorophenyl for HL³_nap], ligands with Ru(H)(CO)(Cl)(PPh₃)₃ afforded exclusively the complexes of composition [(L_nap)Ru(CO)(Cl)(PPh₃)], where N,N,O donor tridentate (L_nap)⁻ was facially coordinated. The ligand 1-{[2-(phenylazo)phenyl]aminomethyl}-2-phenol, HL, 3, was prepared by reducing the aldimine function of HL¹_sal. Reaction of HL with Ru(PPh₃)₃Cl₂ afforded new azosalen complex of Ru(III) in concert with regiospecific oxygenation of phenyl ring of HL. All the new ligands were characterized by analytical and spectroscopic techniques. The complexes were characterized by analytical and spectroscopic techniques and subsequently confirmed by the determination of X-ray structures of selected complexes.     

Effects of increased CO2 concentration and temperature on growth and yield of winter wheat at two levels of nitrogen application

Winter wheat (Triticum aestivum L., cv. Mercia) was grown in chambers under light and temperature conditions similar to the UK field environment for the 1990/1991 growing season at two levels each of atmospheric CO₂ concentration (seasonal means: 361 and 692 μmol mol^?1), temperature (tracking ambient and ambient +4°C) and nitrogen application (equivalent to 87 and 489 kg ha^?1 total N applied). Total dry matter productivity through the season, the maximum number of shoots and final ear number were stimulated by CO₂ enrichment at both levels of the temperature and N treatments. At high N, there was a CO₂-induced stimulation of grain yield (+15%) similar to that for total crop dry mass (+12%), and there was no significant interaction with temperature. This contrasts with other studies, where positive interactions between the effects of increases in temperature and CO₂ have been found. Temperature had a direct, negative effect on yield at both levels of the N and CO₂ treatments. This could be explained by the temperature-dependent shortening of the phenological stages, and therefore, the time available for accumulating resources for grain formation. At high N, there was also a reduction in grain set at ambient +4°C temperature, but the overall negative effect of warmer temperature was greater on the number of grains (-37%) than on yield (-18%), due to a compensating increase in average grain mass. At low N, despite increasing total crop dry mass and the number of ears, elevated CO₂ did not increase grain yield and caused a significant decrease under ambient temperature conditions. This can be explained in terms of a stimulation of early vegetative growth by CO₂ enrichment leading to a reduction in the amount of N available later for the formation and filling of grain.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

Nitrogen rhizodeposition assessed by a NH3 shoot pulse-labelling of Lolium perenne L. grown on soil exposed to 9 years of CO2 enrichment

The effects of elevated CO₂ concentration upon rhizodeposition of nitrogen were investigated on field-grown Lolium perenne planted in soil cores set into the resident soil of a intensively managed ryegrass sward treated with elevated CO₂ for nine consecutive years, under two contrasted N fertilisation regimes (Swiss FACE Experiment). The planted cores were excavated from the ambiant (35 Pa pCO₂) and enriched (60 Pa pCO₂) rings at two dates during the growing season (spring and early autumn). The cores were brought back to the laboratory for a pulse-labelling of ryegrass shoots with ¹⁵NH₃, in order to quantify ¹⁵N-rhizodeposition.A recovery of 10–16% of the total ¹⁵N administred to the plant was recovered in the plant–soil system 48 h after the pulse-labelling; significant amounts of ¹⁵N were released into the soil adhering (adhering soil: AS) to the roots (0.44 μg ¹⁵N g AS⁻¹ and 0.60 μg g AS⁻¹ in the spring and the autumn samplings, respectively).In the spring sampling, there was no effect of atmospheric CO₂ concentration on N rhizodeposition. In the autumn sampling, elevated CO₂ stimulated N rhizodeposition that amounted to 7.2 and 5.2 mg ¹⁵N m⁻², under elevated and ambient CO₂, respectively. Nitrogen rhizodeposition was higher at high N (56 gN m⁻²) than at low N fertilisation (14 gN m⁻²), whatever the sampling date investigated.The mechanisms by which elevated atmospheric CO₂ leads to a stimulation of the net root-released N flux remains to be investigated: was it caused by a higher nitrogen immobilisation by the microbial biomass and a reduced re-assimilation of mineralized N and/or by a stimulation of N efflux from roots? Concomitant to the observed reduction of C rhizodeposition, the stimulation of net N efflux suggests that the quality of root released compounds was modified under elevated CO₂ concentration.     

The phosphatase activity of the plasma membrane Ca pump. Activation by acidic lipids in the absence of Ca increases the apparent affinity for Mg

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.     

Selective electrode for dibenzyl dimethyl amoonium cation as indicator of the membrane potential in biological systems

The electrode sensitive to dibenzyl dimethyl ammonium (DDA⁺), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB^?) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA⁺ solutions ranging between 10^?2 and 3 · 10^?6 M in concentration. High selectivity for DDA⁺ was observed in the presence of various inorganic salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA⁺ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27–44 and 45–62). While they determined the DDA⁺ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA⁺ concentration in the bacterial suspension without any pretreatment. It was also shown that the electrode can measure the DDA⁺ uptake in mitochondria during energization.     

Solid-state and proton NMR characterization of an iron(II) complex of a tridentate, facially coordinating N,N,O donor ligand

Despite the many enzymes that use 2-His-1-carboxylate facial triads to bind iron(II), there are few crystallographically characterized synthetic iron(II) complexes of tridentate ligands that bind through two imidazoles and one carboxylate. We report ¹H NMR characterization of the equilibrium between one such ligand and aqueous Fe²⁺. The formation of 1:1 and 2:1 complexes is evident, but the 1:1 complex is never the exclusive compound in solution. This behavior has not been reported previously for N,N,O ligand-iron(II) complexes. The 2:1 ligand/iron complex crystallizes from solution, and it has been completely characterized including an X-ray crystal structure.     

Solid state conformational study of complexes containing the (o-P-C6H4-CN-κN,P) palladium six-membered chelated ring

The Cambridge Structural Database (CSD) V. 5.23 has been searched for all the structures containing the fragment Pd{o-P-C₆H₄-CN-κ²N,P}. Bond lengths, bond angles and the conformations adopted by the PdPCCCN chelated ring have been studied statistically. It has been found Pd-P and Pd-N distances in the mentioned compounds are, respectively, shorter and longer than the mean value found for these parameters when the CSD was searched irrespective of the ligands. The conformation of the chelated ring has been characterized by means of two torsion angles, and can be described in most cases as a plane containing the P and carbon atoms with Pd and N atoms situated at the same side out of this plane. Molecular mechanics calculations have been employed to justify the conformational preferences found. The calculated strain energy suggests a path for the movement of Pd and N atoms from one side of the PCCC plane to the other through a planar conformation. MM calculations in complex (η³-allyl)-(N-(2-(diphenylphosphino)benzylidene)-4-methoxyphenylamine)-palladium(II) indicate that planar conformation is energetically accessible, and the extent of preorganization for the free ligand in this compound has been evaluated of 74%.     

Inhibition of bovine plasma amine oxidase by superoxide dismutase active Cu(II) complexes

Aqueous Cu²⁺ and Cu(II) complexes of salicylate, lysine, and tyrosine decrease the rate of benzylamine oxidation by bovine plasma amine oxidase. Bissalicylato Cu(II) and Cu²⁺ inhibit non-competitively with respect to benzylamine. Lysine, tyrosine, Cu(EDTA)^2?, Zn²⁺, and Co²⁺ do not inhibit, and erythrocyte Cu, Zn superoxide dismutase shows only slight inhibition of the amine oxidase. The data are most consistent with an inhibitory mechanism involving dismutation of O₂^? by the Cu(II) complexes within a site relatively inaccessible to the enzyme superoxide dismutase. Excess lysine significantly decreases inhibition by the bis-lysine complex of Cu(II).     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.     

Activation of tryptophan 5-monooxygenase by calcium-dependent regulator protein.

Rat brainstem tryptophan 5-monooxygenase was activated about 2-fold by rat brain calcium-dependent regulator (CDR) protein. The activation required both ATP and Mg²⁺ in the presence of low concentrations of Ca²⁺.     

Selective inhibition of L-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium,manganese, and cadmium chloride

AlCl₃, MnCl₂, and CdCl₂ inhibited the rates of accumulation of ¹⁴C] L-glutamate and ³H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC₅₀) of GABA transport were 316 μM, 7.4 mM, and 1.4 mM, respectively. Ca²⁺ (1 mM) enhanced the inhibitory effect of Al³⁺ (IC₅₀ decreased to 149 μM) but antagonized that of Mn²⁺ (IC₅₀ = 10 mM) and Cd²⁺ (IC₅₀ = 2.1 mM). For glutamate transport 1 mM Ca²⁺ changed the IC₅₀ values from 299 to 224 μm for Al³⁺, 7.1 to 10 mM for Mn²⁺, and 2 to 3 mM for Cd²⁺. In contrast, the rates of accumulation of ¹⁴C] 2-deoxy-glucose and ³H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al³⁺, Mn²⁺, and Cd²⁺ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.