At low water activity α-chymotrypsin is more active in an ionic liquid than in non-ionic organic solvents

The kinetics of the -chymotrypsin catalysed transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-butanol and the competing hydrolysis were evaluated at fixed water activity in two ionic liquids and two non-ionic organic solvents. In most respects the four solvents behaved similarly. However, at a water activity of 0.33, higher catalytic activity was observed in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide, than in the other solvents, and at a_w=0.11 catalysis was only observed in this solvent.     

Sequence specificity of the hammerhead ribozyme revisited; the NHH rule.

The sequence specificity of hammerhead ribozyme cleavage has been re-evaluated with respect to the NUH rule. Contrary to previous reports it was found that substrates with GAC triplets were also cleaved. This was established in three different sequence contexts. The rate of cleavage under single turnover conditions was between 3 and 7% that of cleavage 3' of GUC. Specificity of cleavage of substrates containing a central A in the cleavable triplet can be described as NAH, where N can be any nucleotide and H any nucleotide but G. As cleavage 3' of NCH triplets has recently been described, the NUH rule can be reformulated to NHH.     

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.      

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.      

Transient lateral transport of platelet-sized particles in flowing blood suspensions.

Concentration profiles of 2.5 microns latex beads were measured to demonstrate lateral transport of platelet-sized objects in flows of blood suspensions; the flows had equivalent Poiseuille wall shear rates (WSRs) from 250 to 1220 s-1. Each experimental trial began with a steady flow of suspension without beads in a thin-walled capillary tube (219 microns ID; 10.2 microns SD). The tube entrance was then switched to a reservoir containing suspension of equal hematocrit, but with beads, for a short interval of flow at the same WSR. This process established a paraboloidal tongue of labeled suspension with a transient concentration gradient at its surface. The tube and contents were rapidly frozen to fix the suspended particles in flow-determined locations. Segments of frozen tube were collected at distances from the entrance corresponding to 13%, 39%, and 65% of the axial extent of the ideal paraboloidal tongue. Concentration profiles were estimated from distances measured on fluorescence microscope images of cross-cut tube segments. Experiments used tubes either 40 or 50 cm long, suspension hematocrits of 0, 15, or 40%, and bead concentrations in the range of 1.5-2.2 x 10(5)/mm3. Profiles for 0% hematocrit suspension, a dilute, single-component suspension, had features expected in normal diffusive mixing in a flow. Distinctly different profiles and more lateral transport occurred when the suspensions contained red cells; then, all profiles for 13% extent had regions of excess bead concentration near the wall. Suspension flows with 40% hematocrit exhibited the largest amount of lateral transport. A case is made that, to a first approximation, the rate of lateral transport grew linearly with WSR; however, statistical analysis showed that for 40% hematocrit, less lateral transport occurred when the WSR was 250 s-1 or 1220 s-1 than 560 s-1, thus indicating that the rate behavior is more complex.     

An estimated shape function for drift in a platelet-transport model.

Prior work has shown that concentration profiles of platelets in flowing whole blood and of platelet-sized beads in flowing blood suspensions can include near-wall excesses. A model to describe this phenomenon was built about a single-component convective diffusion equation. To incorporate redistribution to preferred sites by shear flows of red cell suspensions, the model used a drift shape function (in addition to the commonly used augmented diffusion coefficient). This paper reports experiments that provide an average concentration profile from which the shape function for that model is calculated; the experiments and shape function are for the particular conditions of 40% hematocrit, platelet-sized latex beads (2.5 microns diameter), tube ID of 217 microns, and a wall shear rate of 555 s-1. Less precise estimates of the shape function obtained from data of previous studies indicate that the shape function is similar for the hematocrit of 15%.     

Activity of hammerhead ribozymes containing non-nucleotidic linkers.

Hammerhead ribozymes were synthesized in which the tetranucleotide loop II was replaced by non-nucleotidic linkers of 7, 13, 17 and 19 atoms length. Ribozymes with 17 and 19 atom linkers, in combination with a 4 base pair stem II, had catalytic efficiencies which were 2 fold increased to that of the parent ribozyme with a tetranucleotide loop. Ribozymes with these linkers, but in combination with a 2 base pair stem II, showed a 2 fold decrease in catalytic efficiency when compared to the parent ribozyme. Prolonged preincubation in the presence of MgCl2 was required for hexaethylene glycol linker-modified ribozymes to obtain maximum activity and reproducible kinetic data.     

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.     

Synthesis of sugar-modified nucleoside 5'-triphosphates with partially purified nucleotide kinases from calf thymus.

The partial purification of some nucleoside monophosphate kinases (ATP:nucleosidemonophosphate phosphotransferases, EC 2.7.4.4) from calf thymus by chromatography on Blue Sepharose to remove interfering phosphatase activity is described. Their specificities towards nucleoside monophosphates modified in the sugar are investigated. Pyrimidine nucleoside monophosphate kinase is not very much affected by such modifications, whereas GMP kinase does not tolerate such alteration. The effect on AMP kinase is intermediate.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.