Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long-range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.     

DNA rearrangements generating artificial promoters

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.     

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.     

Evidence that Fe2+ complexes of 3-aminopicolinate and 3-mercaptopicolinate activate and inhibit phosphoenolpyruvate carboxykinase

3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe²⁺ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe²⁺ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe²⁺ at pH 7.5 was determined to be 2:1 when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe²⁺ complexes. The ratio of 3-mercaptopicolinate or 3-aminopicolinate to Fe²⁺ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:1. In the absence of Fe²⁺, neither pyridine carboxylate altered the activity of the enzyme. These results indicate that the two pyridine carboxylates can interact with phosphoenolpyruvate carboxykinase as Fe²⁺ coordination complexes.     

Interaction of jasmonic acid and blue light in the regulation of arabidopsis morphogenesis

The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses.     

Intrasubunit and intersubunit interactions controlling assembly of active synaptic complexes during Hin-catalyzed DNA recombination

Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Increased -1 ribosomal frameshifting efficiency by yeast prion-like phenotype [PSI+]

Programmed -1 ribosomal frameshifting (-1RF) is usually regulated by a slippery sequence and an RNA secondary structure but can be affected by the slippery sequence combined with translational perturbation. Dramatic increases of -1RF efficiencies arise from the slippery sequences in [PSI+], an epigenetic modifier of translation termination fidelity. Curing of [PSI+] abolished such increases of -1RF efficiency. Enhanced -1RF frequency at the slippery sequences could be another physiological effect induced directly or indirectly by the perturbation of the translation process in [PSI+] cells.     

The state transition mechanism—simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H⁺ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved.     

Relationship between 1-aminocyclopropanecarboxylate malonyltransferase and D-amino acid malonyltransferase

An enzyme preparation isolated from mungbean hypocotyls catalyses the malonyl-CoA-dependent N-malonylation of 1-aminocyclopropane-1-carboxylic acid (ACC), D-phenylalanine (Phe), D-methionine and 2-aminoisobutyric acid with K_m values of 0.15, 0.8, 3.4 and 5.1 mM, respectively L-enantiomers of Phe and methionine were, however, not malonylated by the enzyme preparation. When ACC was tested on D-Phe malonyltransferase activity, or when D-Phe was tested on ACC malonyltransferase activity, these compounds exhibited competitive inhibition kinetics with K_i values similar to their respective K_m values. Such a relationship suggests that malonylations of ACC and D-amino acids are catalysed by the same enzyme. This view was further supported by the observations that the ratio ACC-D-Phe malonyltransferase activities remained constant throughout various fractionation steps and both enzyme activities were inhibited similarly by various sulphydryl reagents and 1-aminocycloalkane-1-carboxylic acids.     

Photoactivation of the manganese catalyst of O2 evolution. I. Biochemical and kinetic aspects

Photosynthetic O₂ evolution requires a Mn complex which is activated by light. An analysis of this activation process yielded the following results:

1.

1. In any given illumination, the time course is first order, the rate being proportional to the number of inactive O₂-evolving System II trapping centers (the quantum yield being invariant).     

Control of choline oxidation in rat kidney mitochondria

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173 ± 64 μM and a Vmax of 0.4 ± 0.1 nmol/min/mg mitochondrial protein (at 10 °C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 °C (125 ± 6 pmolO₂/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96 ± 4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H⁺ ejection/2e^? ratio approaches an average value of 3 when K⁺ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ejection/2e^? ratio of 2 is observed. (3) The low stoichiometry of 3H⁺ ejected (instead of 4) per 2e^? and the high rate of H⁺ back-decay (0.1615 $\text{ln}\text{δ-}\text{(}\text{ngatom}\text{)}\text{H}{}^{\mathrm{+}}\text{s}$ and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K⁺/H⁺ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.     

The roles of intrinsic kinase and of kinase/activator protein in the enhanced phosphorylation of pyruvate dehydrogenase complex in starvation

The calcium ionophore properties of A23187 and of two lysophosphatidic acid (LPA) analogs (1-palmitoyl- and 1-hexadecyl-sn-glycero-3-phosphate or P-GPA and H-GPA, respectively) were compared using platelet membrane vesicles loaded with 45Ca. Half maximal effect (HME) was obtained at 5 microM and 10 microM for H-GPA and P-GPA, respectively, against 0.7 microM for A23187, which released 2 times more Ca. The three compounds also induced platelet aggregation with a HME at 0.5 microM, 0.3 microM and 0.01 microM for A23187, P-GPA and H-GPA, respectively. The clear dissociation between the two effects appearing for both LPA raises some doubt about the general idea that (lyso) PA participate in cell activation through their calcium ionophore properties.     

Interaction between brain enzymes glutamate dehydrogenase and aspartate aminotransferase.

Fluorescence spectroscopic methods were used to study the interaction between aspartate aminotransferase and glutamate dehydrogenase isolated from pig brain. The conversion of the P-pyridoxal form of the aminotransferase to the P-pyridoxamine form of the enzyme is easily monitored by recording emission spectra upon excitation at 330 nm. Evidence for the interaction between the enzymes was obtained from fluroescence measurements conducted on aspartate aminotransferase label with a fluorescence probe (1-5-AEDANS) attached to one sH residue of the protein. The interaction of the aminotransferase (1μM) with glutamate dehydrogenase (2μM) brings about an enhancement as well as a blue shift in the band position of the fluorescence emitted by the dansyl chromophore. Polarization of fluorescence measurements conducted over a wide range of temperatures reveal that the rotational correlation time of aspartate aminotransferase (35 n.seconds) is increased to a value of 100 n.seconds upon addition of glutamate dehydrogenase.     

Biogenesis of chloroplast membranes X. Changes in the photosynthetic specific activity and the relationship between the light harvesting system and photosynthetic electron transfer chain during greening of Chlamydomonas reinhardi y-1 cells

Specific activities of photophosphorylation and light-dependent pH rise at different stages of the greening process, have been measured as a function of the illumination intensity.