Synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP: binding studies using continuous-flow dialysis.

The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme. In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods. The results obtained show that UTP binds to ATCase in the absence of CTP. Nevertheless, this binding does not induce any inhibition unless CTP is present. The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites. However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role. CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold. Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme. These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.     

The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Inferring landscape resistance to gene flow when genetic drift is spatially heterogeneous

In connectivity models, land cover types are assigned cost values characterizing their resistance to species movements. Landscape genetic methods infer these values from the relationship between genetic differentiation and cost distances. The spatial heterogeneity of population sizes, and consequently genetic drift, is rarely included in this inference although it influences genetic differentiation. Similarly, migration rates and population spatial distributions potentially influence this inference. Here, we assessed the reliability of cost value inference under several migration rates, population spatial patterns and degrees of population size heterogeneity. Additionally, we assessed whether considering intra-population variables, here using gravity models, improved the inference when drift is spatially heterogeneous. We simulated several gene flow intensities between populations with varying local sizes and spatial distributions. We then fit gravity models of genetic distances as a function of (i) the ‘true’ cost distances driving simulations or alternative cost distances, and (ii) intra-population variables (population sizes, patch areas). We determined the conditions making the identification of the ‘true’ costs possible and assessed the contribution of intra-population variables to this objective. Overall, the inference ranked cost scenarios reliably in terms of similarity with the ‘true’ scenario (cost distance Mantel correlations), but this ‘true’ scenario rarely provided the best model goodness of fit. Ranking inaccuracies and failures to identify the ‘true’ scenario were more pronounced when migration was very restricted (<4 dispersal events/generation), population sizes were most heterogeneous and some populations were spatially aggregated. In these situations, considering intra-population variables helps identify cost scenarios reliably, thereby improving cost value inference from genetic data.     

Isolation and partial characterization of a new mutant for sterol biotransformation in Mycobacterium sp.

Summary A new sterol biotransforming mutant was isolated from NRRL-B3683 Mycobacterium sp. after nitrosoguanidine mutagenesis. The mutant showed an enhanced ability to biotransform stigmasterol into 17-ketosteroids compared with the parental strain.     

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.     

Zeta potential of protoplasts from gravireacting maize roots

Protoplasts were isolated from cortical cells of the elongating zone of maize (Zea mays L. cv. LG 11) roots and submitted to microelectrophoresis. Significant and transient differences in zeta potential between protoplasts from upper and lower root sides were compared with the gravireaction and the differential elongation of these roots. The maximum difference in the zeta potential was obtained between protoplasts from the upper and lower cortical cells after 90 min, exactly the time of gravipresentation for which the maximum rate of gravireaction was observed. In addition, this almost corresponded to the time for which the difference between the elongation rates of upper and lower sides of the extending zone began to increase. Consequently, the changes in the charges of the plasmalemma of the cortical cells from the growing part of roots could be more or less directly related to the root graviresponse.     

Polyploidy and aspartate-transcarbamylase activity in Hippocrepis comosa L.

The DNA content of plants which were sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of DNA is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes.Abbreviations ATCase aspartate transcarbamylase - CAP carbamylphosphate - EDTA ethylenediaminetetracetic acid - Tris trihydroxymethylaminomethane - AMP adenosine monophosphate - ATP adenosine triphosphate - CMP cytidine monophosphate - CTP cytidine triphosphate - UMP uridine monophosphate - UTP uridine triphosphate