The effect of the amino-acid side chains on the energy profiles for ion transport in the gramicidin A channel

Computations on the energy profiles for Na+ in the gramicidin A (GA) channel have been extended by introducing the effect, previously neglected, of the amino acid side chains of GA, fixed in their most stable conformations. The calculations have been performed in two approximations: 1) with the ethanolamine tail fixed in its most stable conformation, 2) with the tail allowed to optimize its conformation upon the progression of the ion. In both approximations the overall shape of the energy profile is very similar to that obtained in the absence of the side chains. One observes, however, a general lowering of the profile upon the adjunction of the side chains. The analysis of the factors responsible for this energy lowering indicates that it is due essentially to the electrostatic and polarisation components of the interaction which interplay differently, however, in the different parts of the channel. A particular role is attributed in this respect to the tryptophan residues of GA. The role of the 4 tryptophans present, Trp 15, 13, 11 and 9, is individualized by stripping of one of them at a time. The strongest effect on the energy deepening is due to Trp 13 and is particularly prominent in the entrance zone at 14.5A from the center of the channel. The result indicates the possibility of investigating theoretically the effect on the energy profiles of the substitution of the "natural" side chain by others.     

Complete 1H assignments of the non-exchangeable protons of the non self-complementary heptadeoxyribonucleotide d[(GTCGTCA).(TGACGAC)] and its component strands by high field NMR

The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50 degrees C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.     

Inhibition of S-adenosyl-L-methionine sterol-C-24-methyltransferase by analogues of a carbocationic ion high-energy intermediate. Structure activity relationships for C-25 heteroatoms (N, As, S) substituted triterpenoid derivatives

Microsomes from maize seedlings are capable of catalyzing the C-24 alkylation of 4,4,14 alpha-trimethyl-9 beta,19-cyclo-5 alpha-cholest-24-en-3 beta-ol (cycloartenol) by (S)-adenosyl-L-methionine (AdoMet) leading to 24-methylene cycloartanol. Derivatives of cycloartenol bearing a nitrogen atom at C-25 have been previously shown to be potent inhibitors of the AdoMet-cycloartenol-C-24-methyltransferase (Narula, A. S., Rahier, A., Benveniste, P., and Schuber, F. (1981) J. Am. Chem. Soc. 103, 2408-2409). In order to determine the molecular parameters of the inhibition and to gain information about its mechanism, various azasteroids and analogues have been synthesized and assayed. The following results have been obtained. i) The presence of a positive charge at position 25 was found to be the major cause of the inhibition since electrostatically neutral isosteric compounds possessing a carbon in place of the nitrogen atom were not inhibitory. The positive charge leading to inhibition may be conferred by a protonated amine, a quaternary ammonium group, as well as by a sulfonium or an arsonium group. ii) A steroid-like structure of the inhibitor was also important. And iii) the presence of a free 3 beta-hydroxy group and the bent conformation of cycloartenol, which are essential molecular features of the substrate for the methylation reaction, were no longer required to observe inhibition. The data obtained strongly support the idea that C-25 heteroatoms (N, As, and S), substituted triterpenoid derivatives possessing a positive charge at position 25, are analogues of a carbocationic high-energy intermediate involved during the reaction catalyzed by the AdoMet-cycloartenol-C-24-methyltransferase.     

Analysis of the interaction of rabbit skeletal muscle adenylate deaminase with myosin subfragments. A kinetically regulated system

The interaction of rabbit skeletal muscle adenylate deaminase with myosin fragments (heavy meromyosin and subfragment-2) has been studied by analytical centrifugation, gel chromatography, and stopped flow light scattering. Formation of the complex is highly cooperative with respect to addition of two molecules of adenylate deaminase/molecule of myosin fragment to form a ternary complex. Ternary complex formation is also highly pH-dependent with less complex formed at higher pH values, and the pH dependence is steeper with heavy meromyosin than with subfragment-2. At pH 6.5, the dissociation constant for the heavy meromyosin-deaminase complex is approximately 1.2 X 10(-15) M2. Over the pH range 6.5-7.0, rate constants for the formation and dissociation of both the ternary and binary complexes of adenylate deaminase with heavy meromyosin have been determined. From analysis of the time course of stopped flow light scattering, the association steps are found to be extremely rapid, while the rate constant for dissociation of the first molecule of adenylate deaminase from the ternary complex is quite slow. This rate constant increases as the pH increased, but is sufficiently low that the interacting system does not equilibrate on the time scale of mass transport experiments (sedimentation velocity and gel chromatography), and thus displays apparent "slow" behavior. The kinetic regulatory properties of adenylate deaminase are influenced by heavy meromyosin and subfragment-2, particularly with respect to inhibition by GTP. The association and dissociation of adenylate deaminase and myosin fragments and the resultant changes in kinetic properties of the adenylate deaminase can markedly alter the time course of the enzymatic reaction. The time scale over which this interaction is modulated by changes in pH may have significance in the metabolism of exercising muscle.     

The branch point effect. Ultrasensitivity and subsensitivity to metabolic control

The interdependence of the activities of branch point enzymes which compete for a common substrate can yield ultrasensitivity or subsensitivity to control, even if the competing enzymes follow Michaelis-Menten kinetics. The nature of this "branch point effect" for a particular system depends on the kinetic parameters of the competing enzymes, the rate of substrate production leading into the branch point and the type of regulatory mechanism involved. With physiologically reasonable parameter values, the branch point effect can give ultrasensitivity equivalent to an allosteric enzyme with a Hill coefficient of 8 or higher. An experimental example of this ultrasensitivity was provided by the branch point between isocitrate lyase (of the glyoxylate bypass) and isocitrate dehydrogenase in Escherichia coli. The glyoxylate bypass is very active during growth on acetate but its flux decreases by a factor of approximately 150 upon addition of glucose. This inhibition is brought about by two relatively modest events: a 4-fold increase in the maximum velocity of isocitrate dehydrogenase and a factor of 5.5 decrease in the rate of isocitrate production. The mechanism which underlies this sensitivity amplification is discussed.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Derivatives of [3H]serotonin in organ cultures of hamster pineal gland

The purpose of this study was to determine the viability of the hamster pineal gland in organ culture and to test the effect of norepinephrine (NE) on [3H]serotonin derivatives. In this study, elevated levels of melatonin (7-fold, p less than .05), 5- hydroxytrytophol (5-fold, p less than .001), 5-methoxytryptophol (1.78-fold, p less than .05), and depressed levels of 5-hydroxyindoleacetic acid (3.8-fold, p less than .02) and methoxyindoleacetic acid (1.78-fold, p less than .05) were detected in the glands following the addition of NE to the medium. In a separate experiment, melatonin concentration in the media was also periodically measured by radioimmunoassay to determine the viability of the organ culture over a four-day period. The melatonin level on day 2 (2321 +/- 106 pg/gland) was significantly higher (p less than 0.01) than on day 3 (1542 +/- 86 pg/gland) or day 4 (805 +/- 39 pg/gland). The results of these experiments verify the viability of the hamster pineal organ culture and show that the gland responds to NE in vitro.     

Short-wave ultraviolet irradiation increases photo-affinity labeling of benzodiazepine sites

Photoaffinity labeling of benzodiazepine sites with [3H]-flunitrazepam was examined using either long-wave (366 nm) or short-wave (254 nm) ultraviolet irradiation. A multiple exposure protocol was employed so that the time course of the process could be determined as well as the fraction of total sites labeled. At 366 nm, approximately 20% of the total sites present were labeled and the remainder showed reduced affinity for flunitrazepam, in agreement with published reports. When membranes from either cortex or cerebellum were irradiated at 254 nm, however, the fraction of sites labeled increased above 40%. The change in the ratio of labeled sites to those showing reduced affinity, as well as differences in the time courses of the two phenomena, are taken as evidence that the labeling and affinity change are independent processes rather than two effects of a single event. Inhibition of labeling by clonazepam and Ro15-1788 indicated the "central" nature of the labeled sites.