Enzyme activity assessment

Typically, neurotoxic effects of drugs on monoamine neurons have been assessed from reductions in brain levels of monoamines and their metabolites, decreases in the maximal activity of synthetic enzymes activity, and decreases in the active uptake carrier. In the present study, the traditional markers described above have been used, including the measurement of the content of monoamines and their metabolites in brain at several different timepoints following drug administration. Since reports in the literature have documented that MDMA and MDA can inhibit the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis (Stone et al. 1986 Stone et al. 1987). it is unclear whether MDMA-induced reductions in the content of serotonin and its metabolite 5-hydroxyin-doleacetic acid (5-HlAA) may be due to suppressed neurotransmission in otherwise structurally intact serotonin neurons or may represent the eonsequenee of the destruction of serotonin neurons and terminals. 

PBPK and classical pharmacokinetic models both have valid applications in lead risk assessment. Both approaches can incorporate capacity-limited or nonlinear kinetic behavior in parameter estimates. An advantage of classical pharmacokinetic models is that, because the kinetic characteristics of the compartments of which they are composed are not constrained, a best possible fit to empirical data can be arrived at by varying the values of the parameters (O Flaherty 1987). However, such models are not readily extrapolated to other species because the parameters do not have precise physiological correlates. Compartmental models developed to date also do not simulate changes in bone metabolism, tissue volumes, blood flow rates, and enzyme activities associated with pregnancy, adverse nutritional states, aging, or osteoporotic diseases. Therefore, extrapolation of classical compartmental model simulations... 

The desulfinase enzyme from R. erythropolis IGTS8 and R. erythropolis KA2-5-1 have been isolated and characterized [55,126,163,164] however, the latter enzyme had to be cloned into E. coli, to obtain sufficient enzyme for characterization. Both enzymes have an optimum activity at a temperature of 35°C, but slightly different pH optimum (Table 7). The Km values for HBPSi for the two enzymes were similar but the kcat values were different, probably due to the use of different assays to assess enzyme activity. The fccat values for the IGTS8 enzyme did match closely with that reported by the Gray study [53], Expression of the KA2-5-1 enzyme in E. coli required co-expression of chaperonin genes, groEL/groES. 

Measurement of exoenzymatic activities is potentially useful in detecting the effects of toxicants on heterotrophic biofilm communities. Sensitivity and direct relationship with organic matter use and, therefore, microbial growth make extracellular enzyme activities a relevant tool to assess the toxicity of specific compounds. Use of novel approaches that combine enzymatic and microscopic tools (e.g. ELF-phosphatase) may be extremely useful to detect anomalies at the sub-cellular scale. 

The enzymatic activity in soil is mainly of microbial origin, being derived from intracellular, cell-associated or free enzymes. Only enzymatic activity of ecto-enzymes and free enzymes is used for determination of the diversity of enzyme patterns in soil extracts. Enzymes are the direct mediators for biological catabolism of soil organic and mineral components. Thus, these catalysts provide a meaningful assessment of reaction rates for important soil processes. Enzyme activities can be measured as in situ substrate transformation rates or as potential rates if the focus is more qualitative. Enzyme activities are usually determined by a dye reaction followed by a spectrophotometric measurement. 

Castaldi P, Garau G, Melis P (2008) Maturity assessment of compost from municipal solid waste through the study of enzyme activities and water-soluble fractions. Waste Management 28 534-540... 

The presence or absence of pancreatic enzymes can only be satisfactorily decided by intraduodenal intubation and direct examination of samples of small intestinal contents after the administration of a suitable stimulus to pancreatic secretion (Fll). It is not sufficient to look at one enzyme only, such as trypsin, since a specific deficiency of lipase can occur (Sll). Assessment of the degree of hydrolysis of fat in the stools is quite unreliable as a guide to pancreatic enzyme activity (CIO). 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

In the most frequently used method of substrate quantitation, the total amount of change resulting from the presence of the substrate is measured and used to calculate the original amount of substrate present. There must be an adequate enzyme activity to ensure that the conversion of substrate to product takes place in a reasonable period of time, but towards the end of a reaction the concentration of the substrate will be very low and the time taken to reach the equilibrium position will be relatively long. The ratio Vmta/Km is useful in assessing the amount of enzyme that is required and numerical values of about 1.0 are recommended. Provided that the concentration of the substrate is less than the Km value, such conditions will result in a reaction time of just over 3 min for approximately 99% conversion of the substrate. 

Eight human brain tumors contained DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase as assessed by enzyme activity and WB (Rampling et al., 1994). 

Abstract Neuroscientists may wish to quantify an enzyme activity for one of many reasons. In order to do so, the researcher must be able to set up an assay appropriately, and this requires some understanding of the kinetic behavior of the enzyme toward the substrate used. Furthermore, such an understanding is vital if the inhibitory effects of a drug are to be assessed appropriately. This chapter outlines key principles that must be adhered to, and describes basic approaches by which rather complex kinetic data might be obtained, in order that enzyme kinetics and inhibitor kinetics might be studied successfully by the nonexpert. 

Enzyme activity refers to the rate at which a particular enzyme catalyzes the conversion of a particular substrate (or substrates) to one or more products under a given set of conditions. Usually, activity refers to the contribution of many enzyme molecules (often expressed simply as activity per mg of protein or similar) but, in its simplest form, activity refers to the contribution of a single enzyme molecule. The turnover number of an enzyme-substrate combination refers to the number of substrate molecules metabolized in unit time (usually a period of 1 s) under a given set of conditions (see later). These definitions appear, at first glance, to be largely self-explanatory. However, many factors contribute to the final activity of an enzyme, and these must be considered during any assessment of such activity. 

Reversibility of inhibition can be assessed by dialyzing the inhibited enzyme versus buffer, or by passing the inhibited enzyme down a desalting column, or by a dilution procedure. In each case, an uninhibited (control) sample of enzyme should be treated in parallel with the inhibited enzyme sample, as control enzyme activity often changes to some degree as a result of the experimental procedure. 

At the end of the dialysis procedure, the bag is blotted dry and enzyme solution is removed prior to assessment of activity. It may be necessary to include cofactor in the assay mixture if it is possible that a dissociable cofactor was lost from the enzyme during dialysis. As the volume containing enzyme inside the dialysis bag changes to some degree during the dialysis procedure, it is usually necessary to correct measured enzyme activity to reflect this change in volume, and a correction based on protein concentration is often done in this regard. It is normal for activity thus measured to be expressed as a fraction of that in a parallel (dialyzed) control experiment. 

A rapid dilution procedure is routinely used in the author s laboratory to assess reversibility, and it is particularly enlightening if enzyme activity is then determined in a continuous spectrophotometric assay. A microplate assay is set up, in triplicate, as outlined in Table 4-3, later. It is assumed, for the purposes of this example, that the for substrate is 10 tM and that the IC50 for the inhibitor in the presence of 10 tM substrate is 200 tM. The concentration of enzyme used in control wells should be at least tenfold greater than the minimum concentration necessary to catalyze a quantifiable increase in product concentration over the duration of the incubation of substrate with enzyme. 

It is perfectly possible for some substrate-modulator combinations to result in an increase in substrate affinity, an increase in the rate of product formation, or both. The same analytical approaches may be used to study such compounds as have been described earlier to assess inhibitory mechanisms and potencies. However, with an allosteric activator, the dissociation constant might better be termed and values for a and p are more likely to be less than one, and greater than one, respectively. As is the case for inhibition, allosteric enzyme activation would be expected to exhibit substrate dependence (Holt et al., 2004). 

---