Response of plasma IL-6 and its soluble receptors during submaximal exercise to fatigue in sedentary middle-aged men

The pleiotropic cytokine interleukin-6 (IL-6) has been demonstrated to increase during exercise. Little is known regarding the response of the soluble IL-6 receptors (sIL-6R and sgp130) during such exercise. The aim of the current study was to investigate the response of plasma IL-6, sIL-6R and sgp130 during fatiguing submaximal exercise in humans. Twelve participants underwent an incremental exercise test to exhaustion and one week later performed a submaximal exercise bout (96 ± 6% lactate threshold) to volitional exhaustion. Blood samples taken at rest and immediately post exercise were analyzed for IL-6, sIL-6R and sgp130. IL-6 increased (P < 0.01) by 8.4 ± 8.9 pg ml⁻¹ (75.7%) during the exercise period. sIL-6R and sgp130 also increased (P < 0.05) by 2.7 ± 3.9 ng ml⁻¹ (9.6%) and 37.7 ± 55.6 ng ml⁻¹ (9.6%), respectively. The current study is the first investigation to demonstrate that alongside IL-6, acute exercise stress results in an increase in both sIL-6R and sgp130.     

Use of chemical modification in the crystallization of isocitrate lyase from Escherichia coli

Two different crystal forms of isocitrate lyase (ICL) from Escherichia coli have been grown following the chemical modification of the enzyme by either 3-bromopyruvate or ethyl mercuri thiosalicylate (EMTS), contrasting strongly with difficulties in obtaining ordered crystals of the native enzyme. Both crystal forms are obtained using the hanging drop method of vapour diffusion with ammonium sulphate as the precipitant. The crystals diffract well and X-ray photographs of the crystals have established that they are in space groups C222(1) and P3(1) (or its enantiomorph P3(2), respectively. Considerations of the values of Vm and measurements on the crystal density indicate that the asymmetric unit of both crystals contains four subunits.     

Some kinetic properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa.

The steady-state kinetic properties of purified tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa were examined. The results suggest that the enzyme obeys a Rapid-Equilibrium Ordered mechanism, in which phosphoenolpyruvate is the first substrate to bind and 3-deoxy-D-arabino-heptulosonate 7-phosphate is the second product to be released, rather than a Ping Pong mechanism as has been reported previously. The inhibition by tryptophan was found to be parabolic competitive with respect to D-erythrose 4-phosphate and parabolic non-competitive with respect to phosphoenolpyruvate. The enzyme was inactivated by EDTA, and could be protected against this inactivation by phosphoenolpyruvate or 3-deoxy-D-arabino-heptulosonate 7-phosphate but not by D-erythrose 4-phosphate, tryptophan or Pi. This suggests that the enzyme may be a metalloenzyme.     

The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa.

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.     

A new polymorphic probe which defines the region of chromosome 19 containing the myotonic dystrophy locus

The region of human chromosome 19 which includes the myotonic dystrophy locus (DM) has recently been redefined by the tight linkage between it and the gene for muscle-specific creatine kinase (CKMM), which lies just proximal to DM. Utilizing human/hamster hybrid cell lines containing defined breakpoints within this region, we have assigned a number of new probes close to DM. Two of these probes, p134B and p134C, were isolated from a single cosmid clone (D19S51) and detect the same BglI RFLP; p134C detects an additional RFLP with the enzyme PstI. Analysis of these probes in the Centre d'Etude du Polymorphisme Humain families demonstrates tight linkage with a number of markers known to be proximal to DM. A two-point lod score of 6.34 at theta = .025 demonstrates the linkage of this probe to DM. Analysis of a DM individual previously shown to be recombinant for other tightly linked markers indicates that p134C is distal to DM. This result indicates that both the new probe and the existing group of proximal probes including CKMM and ERCC1 probably flank DM and define the genetic interval into which this mutation maps.     

The effect of systematic error on the accuracy of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation (Short Communication)

Systematic errors in initial substrate concentration (s₀), product concentration and reaction time give much larger errors in the Michaelis–Menten parameters unless s₀ is treated as an unknown parameter. These errors are difficult to detect because the fitted curve deviates little from the data. The effect of non-enzymic reaction is also examined.     

Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.     

Binding of bile acids by 100 000g supernatants from rat liver.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.     

An evaluation of ways of using equilibrium dialysis to quantify the binding of ligand to macromolecule.

1. The effect of systematic error (loss of ligand, complex or macromolecule) on three of the experimental designs by which equilibrium dialysis may be used to quantify the interaction of ligand and macromolecule is examined theoretically, and the design that is least sensitive to systematic error is identified. 2. Thirteen methods for fitting the binding isotherm to experimental data are compared by using them to analyse simulated data containing random error, and the most reliable method is identified.