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Kinetic Differences (kinetic + difference)
Selected AbstractsMaintenance of the alcohol dehydrogenase polymorphism in Tiger Salamanders, II.FUNCTIONAL ECOLOGY, Issue 1 2000Differences in biochemical function among allozymes Abstract 1.,Previous studies of Tiger Salamanders demonstrated that variation in alcohol dehydrogenase (Adh) contributed significantly to associations between multilocus heterozygosity and oxygen consumption traits, and that Adh variation was associated with levels of pond-oxygen and metamorphic ability in extreme oxygen environments. Here Adh allozymes are characterized kinetically, and relationships between Adh and oxygen-related physiological traits (ATP/Hb, 2,3-DPG/Hb) are measured. 2.,Kinetic differences were measured among Adh allozymes in the acetaldehyde-to-ethanol direction: kcat/Km ratios (the catalytic constant divided by the Michaelis,Menton constant) were significantly higher in Adh-SF than the other two genotypes, and in Adh-SS compared with Adh-FF. No significant differences were measured in the ethanol to acetaldehyde direction. 3.,Adh-SS had a significantly higher ATP/Hb than Adh-FF, with the Adh-SF intermediate. In addition, a significant interaction between Hb and body mass was measured, such that Adh-FF showed a negative relationship between Hb concentration and body mass while the other two genotypes showed a positive relationship. 4.,These results are consistent with the hypothesis that variation at the Adh locus has adaptive and physiological significance, and that functional differences among Adh allozymes partly explain significant associations between multilocus genotype and organismal traits. [source] Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductaseFEBS JOURNAL, Issue 13 2007Ronnie A. Böck Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80,90%, and D31A and D31L by >,90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in Km for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in kcat/Km. Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in Km; 21-fold decrease in kcat/Km). Only minor variations in the Km for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. [source] Pharmacokinetics of amoxycillin in normal horses and horses with experimental arthritisJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2001J. O. Errecalde The serum and synovial pharmacokinetics of amoxycillin (AMX) were studied after i.v. administration at a dosage of 40 mg/kg to normal horses and horses with induced aseptic carpal arthritis. The best estimates of serum and synovial pharmacokinetic parameters were calculated by mono or bivariable non-linear regression analysis. A biexponential equation was used to describe the concentration vs. time profiles in both normal and arthritic horses. There were no serum kinetic differences between normal and arthritic horses. There were, however, major synovial kinetic changes between these groups. The rate of penetration from serum to synovial fluid was larger in arthritic animals, indicating better penetration in this case. On the other hand, the rate of disappearance from synovial fluid was larger in normal horses, indicating more persistence of the drug in the diseased joint. Synovial AMX availability increased from 21% in normal horses to 79% in arthritic horses. These findings support the use of AMX for the treatment of infectious synovial joint disease produced by susceptible organisms in horses. [source] The ,1 and ,6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kineticsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Janet L. Fisher The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The , family is the largest, with six different subtypes. The ,1 and ,6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the ,1 or ,6 subunit was expressed in mammalian cells along with ,3 and ,2L subunits and the kinetic properties examined with outside-out patch recordings. The ,1,3,2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the ,6,3,2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for ,6,3,2L, compared with the ,1,3,2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the ,1 and ,6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3,TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the ,1 and ,6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition. [source] | ||||||||||