Inhibition of enzymes by metal ion-chelating reagents. Theory and new graphical methods of study

1. The mechanism of inhibition of enzymes by metal ion-chelating reagents is discussed and equations derived. 2. Two distinct mechanisms are postulated and graphical methods are given for differentiating between them. 3. Where the metal ion is actually removed from the enzyme to form a co-ordination complex in solution, a procedure is described for obtaining the stability constant for metal-enzyme interaction, the number of metal ions involved and the stoicheiometry of metal ion-ligand interaction.     

Effect of beta-agonists on expression of calpain and calpastatin activity in skeletal muscle.

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.     

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity.

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A--Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.     

Scaling with body mass of mitochondrial respiration from the white muscle of three phylogenetically, morphologically and behaviorally disparate teleost fishes

White muscle (WM) fibers in many fishes often increase in size from <50 μm in juveniles to >250 μm in adults. This leads to increases in intracellular diffusion distances that may impact the scaling with body mass of muscle metabolism. We have previously found similar negative scaling of aerobic capacity (mitochondrial volume density, V _mt) and the rate of an aerobic process (post-contractile phosphocreatine recovery) in fish WM. In the present study, we examined the scaling with body mass of oxygen consumption rates of isolated mitochondria (VO_2mt) from WM in three species from different families that vary in morphology and behavior: an active, pelagic species (bluefish, Pomatomus saltatrix), a relatively inactive demersal species (black sea bass, Centropristis striata), and a sedentary, benthic species (southern flounder, Paralichthys lethostigma). In contrast to our prior studies, the measurement of respiration in isolated mitochondria is not influenced by the diffusion of oxygen or metabolites. V _mt was measured in WM and in high-density isolates used for VO_2mt measurements. WM V _mt was significantly higher in the bluefish than in the other two species and VO_2mt was independent of body mass when expressed per milligram protein or per milliliter mitochondria. The size-independence of VO_2mt indicates that differences in WM aerobic function result from variation in V _mt and not to changes in VO_2mt. This is consistent with our prior work that indicated that while diffusion constraints influence mitochondrial distribution, the negative scaling of aerobic processes like post-contractile PCr recovery can largely be attributed to the body size dependence of V _mt.     

Modelling the simultaneous processes occurring during the BOD5 test using synthetic waters as a pattern

Synthetic waters imitating BOD₅ test conditions have been studied to model the progress curves of cell growth, organic matter consumption and oxygen demand. Glucose and glutamic acid were used as organic matter and Bacillus subtilis, Escherichia coli and Pseudomonas putida as microorganisms. The concentrations of all the products were measured simultaneously over time and were fitted to the Logistic and Monod models. Following this, the goodness of fit was analysed. Discrimination between models allowed us to conclude that the Logistic model has acceptable accuracy to describe the kinetic behaviour inside a BOD₅ bottle, while the extra parameter in the Monod model would not be statistically justified. Thus, the logistic parameters were determined under different conditions by non-linear regression and their biological meaning was interpreted.     

Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, . . ., 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.