Aquifex aeolicus 3-Deoxy- d - manno -2-Octulosonic Acid 8-Phosphate Synthase: A New Class of KDO 8-P Synthase?

The relationship between 3-deoxy- d - manno 2-octulosonic acid 8-phosphate (KDO 8-P) synthase and 3-deoxy- d - arabino -2-heptulosonic acid 7-phosphate (DAH 7-P) synthase has not been adequately addressed in the literature. Based on recent reports of a metal requiring KDO 8-P synthase and the newly solved X-ray crystal structures of both Escherichia coli KDO 8-P synthase and DAH 7-P synthase, we begin to address the evolutionary kinship between these catalytically similar enzymes. Using a maximum likelihood-based grouping of 29 KDO 8-P synthase sequences, we demonstrate the existence of a new class of KDO 8-P synthase, the members of which we propose to require a metal cofactor for catalysis. Similarly, we hypothesize a class of DAH 7-P synthase that does not have the metal requirement of the heretofore model E. coli enzyme. Based on this information and a careful investigation of the reported X-ray crystal structures, we also propose that KDO 8-P synthase and DAH 7-P synthase are the product of a divergent evolutionary process from a common ancestor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42370/1/239-52-2-205_10520205.pd

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Evidence for long-term averaging of strontium in bovine enamel using TIMS and LA-MC-ICP-MS strontium isotope intra-molar profiles

High spatial resolution micro-sampling of tooth enamel offers the possibility of high temporal resolution isotope data to reconstruct climate, environment, diet and mobility. Questions remain about the duration and pattern of the maturation phase of enamel and the existence and direction of chronological 'time-lines'. LA-MC-ICP-MS measurements of c. 400 μm craters and TIMS analyses of transverse enamel sections of an archaeological bovine third molar were undertaken to investigate the long-term averaging of incorporated strontium. The same gradually increasing isotope profile was obtained from both approaches, indicating that the large increase in spatial resolution did not change the response profile obtained. The results suggest that even at the microscopic scale, strontium is incorporated over a period in excess of 12 months. Averaging of the input signal may result from both long-term retention of strontium in the skeleton and recirculation in the body pool, or long-term maturation of enamel on a microscopic scale. Whichever mechanism is responsible, it may not be possible to recover strontium isotope ratios with a high time resolution from cattle molar enamel unless there is a large imbalance in the amount of strontium supplied by different sources. Consequently, strontium isotope profiles may not be synchronous with those of lighter isotope systems