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At least 289 records · Page 16Linked to original sources
PubMed2003

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.

Amino Acid Sequence↗
PubMed2001

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells.

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.

Animals↗
PubMedDate not supplied

Once a fast cod, always a fast cod: maintenance of performance hierarchies despite changing food availability in cod (Gadus morhua).

To examine whether Atlantic cod maintain constant hierarchies of sprint speeds and muscle metabolic capacities under different feeding regimes, the physiological capacities of individual cod were followed through a starvation-feeding-starvation cycle. We examined sprint speeds and maximal enzyme activities in white-muscle biopsies at each period. We measured the glycolytic enzymes, phosphofructokinase (PFK) and lactate dehydrogenase (LDH), the mitochondrial enzyme, cytochrome C oxidase (CCO), and the biosynthetic enzyme, nucleotide diphosphate kinase (NDPK). Sprint speeds were measured in a laser diode/photocell-timed raceway. As expected, the feeding regime had a marked impact on the physiological capacities of cod, but the responses differed for sprint-swimming and muscle metabolic capacities. The different enzyme activities as well the condition index generally decreased during the first starvation, improved with feeding, and fell again during the second starvation. In contrast, sprint performance improved after feeding but did not fall with the second starvation. Although both the enzyme activities and the sprint speeds showed considerable interindividual variation, sprint speeds were not significantly correlated with the enzyme activities. The hierarchy of sprint performance of the cod was maintained, regardless of the preceding feeding regime, whereas those of muscle metabolic capacities were not.

Animal Feed↗
PubMed1997

How fast is fast enough for clinical laboratory turnaround time? Measurement of the interval between result entry and inquiries for reports.

We measured the time interval from result entry by the clinical laboratory to inquiry for reports by clinicians as a proxy for the actual turnaround time required to meet current patient care needs and to determine whether different patterns of report inquiry occur among clinical departments. The study included 4,004 complete blood cell (CBC) count reports that were sought by the clinical services using the hospital information system. The median time to report inquiry was 90 minutes for routine inpatient tests, 35 minutes for stat inpatient tests, and 30 minutes for the stat outpatient CBC counts. Most reports (range, 86%-94%) from these three subgroups were requested within 4 hours from entry of the results in the hospital information system. Of the routine outpatient test reports, 14%, 23%, and 31% were requested within the first 2 hours, 4 hours, and 8 hours, respectively. Although the interdepartmental variations in the median time for report inquiry were statistically significant for routine inpatient tests, stat inpatient tests, and stat outpatient tests, inquiries for the preponderance of reports for all three groups were within the first 1.5 to 3 hours from entry of the results in the hospital information system. We conclude that the majority of CBC counts and other tests with effects on immediate patient care management must be performed very rapidly on site and cannot be moved off site without compromising current standards of medical practice.

Blood Cell Count↗