Search

Lists titles of research papers on biochemistry. "Redox control of catalysis in ATP-citrate lyase from rat liver"; "Construction and molecular dynamics of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography"; "Studies on the variants of the protein toxins ricin and abrin".

The article presents information about the upcoming papers related to biochemistry that will appear in the 1987 issue of the journal "European Journal of Biochemistry." Some of the papers are "Influence of Different S'-Flanking Sequences of tRNA Genes on Their in Vivo Transcription Efficiencies in Saccharomyces Cerevisiae," by researchers T. Dingermann, K. Nerke and R. Marschatek, "Transcription Factois Mediate rRNA Synthesis During Myogenesis," by researchers P. Zahradka and B. H. Sells, "Influence of the Piotonation Degree on the Self-Association Properties of Adenosine 5'-Triphosphate (ATP)," by researchers R. Tribolet and H. Sigel.

Lists the papers that will be published in the forthcoming issues of the "European Journal of Biochemistry". "The transmembrane arrangement of the ADP/ATP carrier as elucidated by the lysine reagent pyridoxal 5-phosphate," by W. Bogner, H. Aquila, M. Klingenber; "Mammalian aldolases are isomer-selective high-affinity inositol polyphosphate binders," by B. Koppitz, F. Vogel, G. W. Mayr.

This article presents information on forthcoming papers to be published in the "European Journal of Biochemistry." Some of the articles included in the list are, "The Transmembrane Arrangement of the ADP/ATP Carrier As Elucidated by the Lysine Reagent Pyridoxal 5-Phosphate," by W. Bogner, H. Aquila and M. Klingenberg, "Mammalian Aldolases Are Isomer-Selective High-Affinity Inositol Polyphosphate Binders," by B. Koppitz, F. Vogel and G.W. Mayr, "Kinetics of Cadmium and Terbium Dissociation From Calmodulin and Its Tryptic Fragments," by S.R. Martin, S. Linse, P.M.. Bayley and S. Forsen.

The purification and the amino acid sequence of the peptide containing the essential sulphydryl group of arginine kinase from Homarus vulgaris muscle is described. The fractionation of the tryptic digest of arginine kinase labelled with N-[1-14C]ethylmaleimide has been caused out using gel filtration, ion-exchange chromatography and paper chromatography. The composition of the radioactive peptide, isolated from the digest, is reported together with its amino acid sequence. A structural comparison is made between the peptide of arginine kinase and the corresponding active-cysteine containing peptide isolated from rabbit muscle creatine kinase. [ABSTRACT FROM AUTHOR]

Extracellular ATP triggers catecholamine secretion from PC12 cells by activating ionotropic purine receptors. Repeated stimulation by ATP leads to habituation of the secretory response. In this paper, we use amperometric detection to monitor the habituation of PC12 cells to multiple stimulations of ATP or its agonist. Cells habituate to 30 µmATP slower than they do to 300 or 600 µmATP. Modifying external Mg2+ affects the response of cells to 30 µmATP, but does not affect habituation, suggesting that habituation does not necessarily correspond to either stimulus intensity or cellular response. Mg2+ affects the initial response of PC12 cells to 2MeSATP in a manner similar to ATP. Increasing external[Mg2+] to 3.0 mm, however, eliminates habituation to 2MeSATP. This habituation can be partially restored by costimulation with 100 µmUTP. Background application of UTP increases habituation to both ATP and 2MeSATP. This suggests that ATP-sensitive metabotropic (P2Y) receptors play a role in the habituation process. Finally, although Ca2+ influx through voltage-operated calcium channels does not appear to contribute to secretion during ATP stimulation, blocking these channels with nicardipine increases habituation. This suggests a role for voltage-operated calcium channels in the habituation process. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

P-glycoprotein is an ATP-dependent plasma membrane multidrug transporter of broad specificity. A common chemical property of its substrates is that all are lipophilic. Using Hoechst 33342 as the substrate, we have previously shown that P-glycoprotein extracts the substrate directly from the lipid bilayer [Shapiro, A. B., Corder, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 115-121]. In this paper, we determined the leaflet of the plasma membrane from which P-glycoprotein extracts Hoechst 33342. The initial rate of Hoechst 33342 transport upon ATP addition to P-glycoprotein-rich inside-out plasma membrane vesicles decreased slightly with the amount of time previously elapsed for slow diffusion of Hoechst 33342 to the extracellular leaflet. This result is consistent with transport from the cytoplasmic leaflet. Fluorescence resonance energy transfer from donor Hoechst 33342 to acceptor 2-[6-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero-3-phosphocholine (Nbd-C6,-HPC) in the cytoplasmic leaflet was used to monitor the amount of Hoechst 33342 in the cytoplasmic leaflet versus time. The initial rate of decrease of the energy-transfer-related Nbd-C6-HPC fluorescence after ATP addition exceeded that of the Hoechst 33342 fluorescence and continued to decrease after decrease of the Hoechst 33342 fluorescence had ceased. These effects were consistent with transport of Hoechst 33342 from the cytoplasmic leaflet to the aqueous interior of the vesicles, followed by rebinding to the extracellular leaflet. This demonstrates that P-glycoprotein transports drugs from the cytoplasmic leaflet of the plasma membrane directly to the aqueous extracellular medium. This finding has implications for efforts to localize the drug-binding site(s) within P glycoprotein. [ABSTRACT FROM AUTHOR]

Escherichia coli K 12 appears to behave as an enzyme. We show in the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-bisphosphate. Although both phosphofructokinases of E. coli K 12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo. [ABSTRACT FROM AUTHOR]

The calcium pump of sarcoplasmic reticulum possesses high-affinity calcium-binding and ATP-binding sites. At 0°C pH 6.8 and in the absence of calcium, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 μM. In the presence of Ca2+. ATP phosphorylates the enzyme at a much lower concentration: K½ = 100 nM. In the absence of ATP the calcium ions reversibly bind to the high-affinity calcium sites (6.5 nmol/mg); however the following is shown in this paper. 1. Phosphorylation of the enzyme in the presence of calcium leads to the immediate occlusion of the calcium ions bound to the high-affinity sites. 2. Two moles of calcium are occluded per mole of phosphoenzyme formed. 3. Occlusion can be reversed by ADP. 4. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of magnesium reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phosphoenzyme. [ABSTRACT FROM AUTHOR]

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its γ-phosphoryl group to carbamoyl phosphate. This bound ATPB can react with NH3, HCO3- and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO3- are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10-30 µM ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t½ 8-16 s, 22 °C), independent of ATP concentration and little affected by NH3, HCO3- or by incubation of the enzyme with unlabelled ATP prior to the pulse of [γ-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO3- when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO3- (< 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO3- in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 µM) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine (Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321 - 329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent the conformational change postulated by others from steadystate kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO3- -dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase. [ABSTRACT FROM AUTHOR]

The DNA-stimulated 75 000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180 000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme · DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells. [ABSTRACT FROM AUTHOR]

The antibiotic, chlorothricin, inhibits the reaction catalyzed by pyruvate carboxylase from Bacillus stearothermophilus. With steady-state kinetic measurements the following effects can be observed. (a) Inhibition of the overall reaction is competitive with respect to CoASAc, the allosteric activator of this enzyme, and non-competitive with respect to both MgATP and pyruvate (substrates). (b) Chlorothricin raises the Hill coefficient, n, of CoASAc from 2 to nearly 3. On the other hand, interaction of the antibiotic with enzyme is characterized by n = 3. (c) The Michaelis-Menten-type kinetic of MgATP is changed to a kinetic showing substrate inhibition, while the substrate activation of pyruvate carboxylase by pyruvate is suppressed upon the addition of chlorothricin. The data presented in this paper strongly suggest that the conformation of site 1 of pyruvate carboxylase responsible for the regulation of the overall enzyme activity is influenced by chlorothricin and CoASAc in an antagonistic manner. A model is proposed which assumes the existence of hybrid enzyme forms. [ABSTRACT FROM AUTHOR]

Binding of tryptophan, tryptamine and ATP to tryptophanyl-tRNA synthetase was studied by equilibrium dialysis experiments. There arc two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could he measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active direct into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer-monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state. [ABSTRACT FROM AUTHOR]

Recently, Sowadski and colleagues [Knighton, D. R., Zheng. J., Eyck, L. E T., Ashford, V. A., Xuong, N., Taylor, S. S. & Sowadski, J. M. (1991) Science 407. 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase). MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions, of MgATP with numerous conserved residues at the active site defined [Zheng. J., Knighton, D. R., Eyck, L. E T., Karlsson, R., Xuong. N., .Taylor, S. S. & Sowadski, J. M. (1993) Biochemistry. in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the α, β and γ phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [γ-12P]adenosine.5'-(γ-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its γ phosphate. In the presence of 20 μM ATP. adenosine-5'-(γ-4-aminophenyl)triphosphate exhibited apparent K1 values of 103.6. 75.18, 176.28 and 120.00 μM against cyclic A kinase, mitogen-activated protein kinase (p42mapk), mitogen-activated protein kinase kinase and p60c-src, respectively. To illustrate the effectiveness of adenosine-5'-(γ-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity. [ABSTRACT FROM AUTHOR]

The inducible glutamate uptake system in Corynebacterium glutamicum (Krämer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal)was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed. The uptake of glutamate resembles a primary active, ATP-dependent transport mechanism in several respects. (a) The substrate affinity is very high {I.3 µM). (b) Accumulation of glutamate reaches values of greater than 2 · 105, at least as high as those reported for binding-protein-dependent systems in Gram-negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers {carbonyl m-chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4- 5 mM ATP. [ABSTRACT FROM AUTHOR]

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymema cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubules — 22s-dyein complex is dissociated by the binding of ATP to the enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5’-[α-thio]triphosphate (ATP[αS]), Sp and Rp, by light-scattering stopped-flow methods. The dissociation by (Sp)ATP[αS] was rapid and the rate of the light-scattering change by (Sp)ATP[αS] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (Rp )ATP[αS] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s-1). The adenosine 5’-[β-thio]triphosphate (ATP[βS]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dyein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol · mg protein-1 · min-1)/apparent Km (µM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[αS] (Sp or Rp), ATP[βS] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at β phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[αS] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[αS] was one‐quarter to one‐half of that produced by ATP reactivation. [ABSTRACT FROM AUTHOR]

Treatment of Ehrlich ascites tumor cells with 2-difluoromethylornithine (F2MeOrn), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, resulted in depleted putrescine and spermidine content, and reduced growth rate. We have previously shown that adenine ribonucleotide levels are substantially increased in these polyamine-depleted cells. The present paper addresses the question whether the elevated ATP pool is accompanied by a concomitant increase in the dATP pool. if this is the case, the observed growth inhibition could be explained by the well-known dATP-mediated feedback inhibition of ribonucleotide reductase. We found that dNTP pools were not unbalanced and that dNTP synthesis was not arrested in polyamine-depleted cells. Moreover, the dNTP content and the activity of ribonucleotide reductase (CDP reduction) and thymidylate synthase, remained elevated despite the fact that the cells were inhibited in their growth by F2MeOrn treatment. Incorporation of a radiolabeled precursor into DNA was initially lower in F2MeOrn-treated cells than in control cells. However, while incorporation of a radiolabeled precursor into DNA decreased markedly in plateau-phase control cells, it remained at a higher level in cells inhibited in growth by polyamine depletion. This discrepancy may be explained by the fact that polyamine-depleted cells accumulated in the S phase, and that they had an increased content of acid-soluble radiolabeled DNA precursor. Our data indicate that polyamine depletion adversely affects the DNA synthetic machinery by reducing the rate of elongation. [ABSTRACT FROM AUTHOR]

This paper describes a large-scale purification of guanylate kinase (ATP + GMP ⇌ ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X-ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences. 1. Guanylate kinase was purified in five steps to homogeneity: crude extract, ion-exchange chromatography, affinity chromatography and gel filtration twice. 2, The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 × 200 × 150 µm³. Preliminary X-ray results are given. 3, The final sequence shows 186 amino acids (Mr = 20 548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N-terminal blocking group was identified by ¹H-NMR spectroscopy. 4. Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases. [ABSTRACT FROM AUTHOR]

In mammals, carbamoyl phosphate for utilization in pyrimidine biosynthesis is synthesized by a glutamine-dependent carbamoyl-phosphate synthase II which is subject to regulation by 5-phospho-α-D-ribosyl 1-diphosphate (PRib-PP), a positive effector, and MgUTP, a negative effector [Mori, M., Ishida, H. and Talibana, M. (1975) Biochemistry 14, 2622-2630]. We have found that Lineweaver-Burk plots of carbamoyl phosphate synthase activity versus 1/[MgATP] are described by a velocity equation which is a ratio of quadratic polynomials, consistent with a positive homotropic interaction between two catalytic sites for the binding of MgATP (Ks = 16.6 ± 3.1 mM, interaction factor a = 0.00538 ± 0.00245). The activating effect of PRib-PP upon carbamoyl-phosphate synthase is consistent with PRib-PP binding at an allosteric site (Ka = 31.4 ± 6.4 μM) and promoting the binding of a first molecule of MgATP as substrate (interaction factor l = 0.0437 ± 0.0063). Thus MgATP and PRib-PP bind to the E · MgATP complex with respective dissociation constants of a · Ks = 0.089 mM and l · Ka = 1.4 μM while MgATP binds to the E. PRib-PP complex with a dissociation constant of l · Ks = 0.73 mM. Data for the inhibitory effect of MgUTP upon carbamoyl-phosphate synthase indicate that MgUTP competes with MgATP for binding at the catalytic site (Ki = 0.203 ± 0.016 mM). A computer model has recently been developed which enables quantitative simulation of the time-dependent effects of blockade of the pyrimidine pathway by a tight-binding enzyme inhibitor [Duggleby, R. G. and Christopherson, R. I. (1984) Eur. J. Biochem. 143, 221–226]. The velocity equation derived m the present paper provides a quantitative basis for predicting changes in the flux through the de novo pyrimidine pathway m growing cells. [ABSTRACT FROM AUTHOR]

1. The paper analyzes the relationship between membrane potential (Δψ),steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of Δψ results in net Ca2+ uptake leading to all increased steady state pCao. On the other hand, a more marked depression of c results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between Δψ and pCao, in that pCao increases with the increase of Δψ up to a value of about 130 mV, whereas a further increase of Δψ above 130 mV results in a decrease of pCao The phenomenom of Ca2+ uptake following a depression of Δψ is independent of the tool used to affect Δψ whether by inward K+ current via valinomycin, or by inward H+ current through protonopbores or through F1-ATP synthase, or by restriction of e flow. 2.The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady stale and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of Δψ is observed, in that the rate of Ca2+ efflux increases with the value of Δψ. The pCa0 is linearly related to the rate of Ca2+ efflux. 3. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondfia, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. 4. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, Δψ-controlled pathway and that changes of Δψ during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the Δψ-controlled Ca2+ efflux pathway. [ABSTRACT FROM AUTHOR]

In this paper a study is presented of the interaction of N,N′-dicyclohexylcarbodiimide (DCCD) with the H+-ATPase of mitochondria. Inhibition of proton conduction by the membrane sector (F0) of the H+-ATPase has been characterized by kinetics analysis of the effect of DCCD on the anaerobic decay of δμH+ set up by respiration in submitochondrial particles containing (ESMP) or deprived (USMP) of the catalytic sector (F1). The inhibitory effect of DCCD was directly related to the amount of DCCD bound independently of the time of incubation and of the concentration applied. Both in ESMP or USMP 2–3 nmol DCCD/mol F0 were required for the maximal inhibition of proton conduction. In ESMP the anaerobic relaxation of δμH+ is biphasic. Both phases were inhibited by DCCD and the titration curve indicated that the slow phase is directly related to the hydrolytic activity of H+-ATPase. In USMP the anaerobic relaxation of δμH+ is monophasic. DCCD, at low concentrations, restored the biphasic pattern with the appearance of a transient fast phase having a kinetic constant higher than that of the control. Whilst the slow phase was immediately inhibited by DCCD the fast phase started to be inhibited only at concentrations higher than 1 nmol DCCD bound/mg protein. The data reported support the view that the fast phase of proton conduction reflects a conformational state of F0, with higher proton conductivity, which is induced by high aerobic δμH+. Electrophoretic analysis of the labeling by [14C]DCCD of membrane polypeptides indicated that the inhibition of the state of low proton conductivity of F0 can be correlated with binding to the 8000-Mr form of DCCD-binding proteolipid, whilst inhibition of the state of high proton conductivity of F0 can be correlated with binding to the 16000-Mr polypeptide. [ABSTRACT FROM AUTHOR]

The effect of trypsin treatment with respect to the allosteric properties of yeast phosphofructokinase as well as its molecular weight has been studied. In the presence of fructose 6-phosphate trypsin desensitizes yeast phosphofructokinase to ATP inhibition. Under the same conditions, activation of this enzyme by the positive allosteric effector AMP, however, remains uneffected. Addition of MgATP instead of fructose 6-phosphate to the incubation medium containing trypsin protects the enzyme against ATP-desensitization. After prolonged incubation of yeast phosphofructokinase with trypsin the enzyme becomes inactivated both with fructose-6-phosphate and with MgATP. Using density gradient centrifugation sucrose media the effects of trypsin treatment on the sedimentation behaviour of yeast phosphofructokinase has also been investigated. With fructose 6-phosphate, trypsin converts the 560000 form of yeast phosphofructokinase into two enzymatically active forms of 510000 and 350000 daltons respectively, which are both e to ATP inhibition. On the other hand, in the presence of MgATP the peak of enzymatic activity sediments with 160000 daltons. This molecular form has the same high sensitivity to ATP inhibition as the untreated enzyme of molecular weight 560000. After the addition of fructose 6-phosphate, the 160000-molecule seems to dimerize giving an ATP-sensitive form with a molecular weight of 340000. In the light of studies about the existence of several interconvertible forms of yeast phosphofructokinase published recently by our laboratory, the results described in this paper are discussed in terms of the existence of dimeric and trimeric states, regarding the 16000-moiety as the trypsin modified monomer of the enzyme. Kinetic and allosteric properties of these states including conditions for their formation and stabilization are summarized and the peculiarities of the trypsin-unmodified and trypsin-modified enzyme are compared. [ABSTRACT FROM AUTHOR]