Biochimica et biophysica acta最新文献

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

Plectonema boryanum shows temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. Low temperature fluorescence studies have shown that in vivo the nitrogen fixing and photosynthesizing cells are adapted to 'state 2' and 'state 1', respectively. During nitrogen fixation phycobilisomes seem to transfer excitation energy to photosystem I whereas during oxygenic photosynthesis the energy is transferred to photosystem II. The state 2 adapted N-phase cells failed to undergo transition to state 1 while P-phase cells exhibited state 1 to state 2 transition. The nitrogen fixing cells showed a decreased level of psbC transcript, lack of CP47 in thylakoid membrane, and presence of the F685 peak but absence of the F695 peak in 77 K fluorescence spectra. These results suggest that the metabolic and molecular changes associated with nitrogen fixation may favor direct energy transfer from the phycobilisomes to photosystem I. This should help the organism to achieve low photosystem II and high photosystem I activity to set temporal separation of nitrogen fixation and photosynthesis for photoautotrophic growth under diazotrophic conditions.

Plectonema boryanum shows temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. Low temperature fluorescence studies have shown that in vivo the nitrogen fixing and photosynthesizing cells are adapted to 'state 2' and 'state 1', respectively. During nitrogen fixation phycobilisomes seem to transfer excitation energy to photosystem I whereas during oxygenic photosynthesis the energy is transferred to photosystem II. The state 2 adapted N-phase cells failed to undergo transition to state 1 while P-phase cells exhibited state 1 to state 2 transition. The nitrogen fixing cells showed a decreased level ofpsbC transcript, lack of CP47 in thylakoid membrane, and presence of the F685 peak but absence of the F695 peak in 77 K fluorescence spectra. These results suggest that the metabolic and molecular changes associated with nitrogen fixation may favor direct energy transfer from the phycobilisomes to photosystem I. This should help the organism to achieve low photosystem II and high photosystem I activity to set temporal separation of nitrogen fixation and photosynthesis for photoautotrophic growth under diazotrophic conditions.

After isolation and purification, the H(+)-ATPase from chloroplasts, CF(0)F(1), contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]ADP leads to tight binding of azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF(0)F(1). Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF(0)F(1). The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF(0)F(1) and with non-derivatised CF(0)F(1). ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

NMR spectroscopy was used to characterize the binding properties of polyamines to Escherichia coli tRNA. The (15)N NMR spectra of three (15)N-enriched N-substituted putrescine derivatives (DMP, DEP and DBP) were recorded in the presence of tRNA, and the spin relaxation times of the nitrogen nuclei were measured. From these data, the activation parameters for the rotational correlation times of the (15)N nuclei were determined. The present data indicate that the nature of the amino substituents does play a relevant role in controlling the polyamine-tRNA interaction. This study also provides a rationale for the in vivo antiproliferative effect of DBP against tumoral cells.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

The surface tension behaviour of oxidised cytochrome c (cyt c) solution was characterised at various pH and ionic strength at the air/water interface. The pendant drop method employing digital image analysis of the drop shape was applied to the measurement of the surface tension. The adsorption properties of cyt c were utilised to study the protein conformation change effected by acidification and ionic strength. At high ionic strength, the saturated steady-state surface tension shows a cooperative change centred around 3.6 induced by a decrease in pH. Using spectroscopic experiments, the apparent pK of the acid-induced transition of horse cyt c from the native to the molten globular state is equal to 3.5. This fact indicates that the saturated steady-state surface tension is a parameter which might be used to monitor conformation changes of cyt c.

The effect of perfusion with elevated glucose concentrations on hypoxic myocardium was investigated in isolated Langendorff guinea pig hearts. For that purpose, mechanical (heart rate, systolic peak pressure and coronary flow) and electrophysiological (monophasic action potential duration=MAP, ectopic beats) data were evaluated. At the end of the experiments the hearts were examined histologically after trypan blue vital staining for quantification of irreversible myocardial damage. In the absence of insulin moderate glucose elevation (from 5 to 15 mM) exerted beneficial effects on hypoxic hearts: the depressed contraction was improved, the action potential shortening partly reversed and the percentage of irreversibly damaged myocytes diminished. Glucose did not have any effect on heart rate and arrhythmias under hypoxia or reperfusion. A contribution of cardiac ATP-dependent K+ channels to the effects of glucose could be excluded by further experiments. Thus, blocking these channels with high glibenclamide concentrations did not affect the action of glucose on MAP and contraction. To some degree the glucose effect on MAP, but not on systolic pressure, was also observable under normoxic conditions.

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.