Enzyme activity measurement, effect

Following work by the same group addressed some of the major problems arising when electrochemical biosensors are in contact with food matrices pH effect and particle effect. Both problems were solved treating the biosensor surface with a Tween20 /phosphate buffer solution (pH 7.5) after the incubation with pesticide. The treatment was successful in removing the particulate, the correct pH for enzyme activity measurement was attained and the pesticide enzyme inhibition... 

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

Of the various factors suggested to play a part in impaired adipocyte function in insuhn resistance and NIDDM, increased TNF-a expression and production have attracted interest. TNF-a is produced and secreted from adipose tissue in obesity and thus acts in an autocrine fashion to alter adipocyte function during obesity-hnked insuhn resistance [385-387]. Long-term exposure (>6h) to TNF-a has been shown to stimulate hpolysis in adipocytes [368, 388], despite inducing reduced HSL expression [389, 390]. An early study showed a down-regulation of HSL gene expression upon TNF-a treatment of 3T3-L1 cells, as measured by Northern blot analysis and enzyme activity measurements [391]. A similar effect, although much more moderate, was seen more recenfly at the protein level [264]. In a study wifh primary rat adipocytes, however, no alteration of the levels of HSL protein occurred upon treatment wifh TNF-a [392]. 

As is the case for most enzyme activities measured in vitro, there is some doubt whether the histidine decarboxylase activities determined as above in various organs truly reflect the contribution of these organs to histidine decarboxylation in the intact animal. In vivo measurements give an overall picture of histidine decarboxylation in the living animal, but they can give little indication of the contribution made by individual organs. Moreover, the interpretation of such measurements is rendered difficult by bacterial decarboxylation of histidine in the gut, by metabolic destruction of histamine, and by the release of histamine from storage sites. Nevertheless, such measurements have provided much useful information, and they are particularly suited to the study of the effectiveness of histidine decarboxylase inhibitors in intact animals. As with in vitro methods the in vivo measurements can, in theory, be made either on the carbon dioxide or on the histamine formed in the decarboxylation. 

The effect of Tween 20 loading on enzymatic hydrolysis of pretreated CWR was studied at five different loading levels, including 0, 0.05, 0.1, 0.15, and 0.2 g Tween 20/g-dry solid. At the end of 72 h hydrolysis, a 1-ml aliquot was withdrawn for sugars measurement. At the same time, another 1-ml aliquot was withdrawn for protein and enzyme activity measurement. For enzyme protein concentration and activity measurements, the sample pretreatment procedures before measurement were the same as described in Effect of Tween 20 and BSA on Enzyme Protein Concentration and Activity. ... 

Clinically, the main area of concern is with a decrease in enzyme levels, usually manifested as a decrease in the enzyme activity measured in vitro. Such decreases in enzyme activity can arise from a variety of factors, either by causing a reduction in concentration of the enzyme or by directly affecting its action (usually by inhibition). There are occasions when an increase in activity or concentration is observed that although uncommon, will have an effect on the drug metabolizing properties of the enzyme. The following arc some of the factors leading to a decrease in activity ... 

Figure 4 shows the remaining enzyme activities measured in all three experiments. Despite the different levels of inhibition, all experiments show the successful detection of sarin with respect to a drop in absorbance. Experiment 2 shows clearly that the sample cleanup absorbent packed in the device was effective in removing the Fluoride ions, and more then 50% of the immobihzed enzyme was inhibited. The results show that on-chip sample preparation was even better than the conventional process outside the LOC device. 

Chemical Pathology. Also referred to as clinical chemistry, this monitoring procedure involves the measurement of the concentration of certain materials in the blood, or of certain enzyme activities in semm or plasma. A variety of methods exist that allow (to variable degrees of specificity) the definition of a particular organ or tissue injury, the nature of the injurious process, and the severity of the effect (76). 

FIGURE 23.12 Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 /xM AMP. In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in fiM) are indicated above each curve, (c) The effect of AMP (0, 10, and 25 fiM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphos-phate. Activity was measured in the presence of 10 /xM fructose-1,6-bisphosphate. 

A bioassay is a test designed to measure the effect of a chemical on a test population of organisms. The effect may be a physiological or biochemical parameter, such as growth rate, respiration, or enzyme activity. In the case of drilling fluids, bioassays lethality is the measured effect. 

Trichloroethylene may occur in drinking water along with other chlorinated hydrocarbons, so effects of these chemicals in combination are of interest to public health. Hepatotoxicity, as measured by plasma enzyme activity, was increased synergistically in rats by oral administration of carbon tetrachloride combined with trichloroethylene (Borzelleca et al. 1990). In addition, synergistic effects were implicated in a 3-day study in... 

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. 

In contrast, following a treatment regimen of 20 mg/kg MDMA, there were no significant differences in the density of [3H]mazindol-labeled norepinephrine (NE) uptake sites (fmol/mg protein) in the frontal cerebral cortex between saline-treated (159 17) and MDMA-treated (152 5) animals. With respect to the dose of MDMA, serotonin levels appeared to be more readily decreased (45 percent reduction at 5 mg/kg), while comparable reductions in 5-HlAA levels and serotonin uptake sites were noted only at 10 or 20 mg/kg MDMA. This apparent discrepancy among the three serotonergic markers measured in the present study may relate to effects of lower doses of MDMA on synthetic enzyme activity (i.e., TPH), whereas the effects of higher doses of MDMA in reducing all three markers may relate in part to effects on TPH activity and in part to destruction of serotonin neurons as evidenced by decreases in serotonin uptake sites. 

Again, if we wish to measure the effects of an inhibitor on enzyme activity, we must cast Equation (A2.16) in terms of reaction velocity. Combining Equation (A2.13) with Equation (A2.16), we obtain... 

Dihydroxybenzoic acid (DHB) is also a commonly used tool to measure the pharmacological effects of HIF-la stabilization via PHD inhibition. Recently, it was shown that mice pretreated with DHB (100 mg/kg, i.p.) showed a marked resistance to the neurotoxic effects of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) via protection of dopaminergic cell loss and striatal denervation. Importantly, this protection was seen to coincide with HIF-la stabilization, and the prevention of the MPTP-induced loss of ferroportin and striatal iron. Additionally, in these studies, DHB was also observed to block MPTP-induced reduction in mitochondrial pyruvate dehydrogenase, at both the mRNA level and through the measurement of enzyme activity in midbrain substantia nigra [26]. 

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