Other Enzyme Measurements

OTHER ENZYME MEASUREMENTS 2.5.1 Tissue Cytochromes P450... 

Neither of these enzymes (see Chapter 2) is widely used for the diagnosis of hepatotoxicity because, unlike the other enzyme measurements mentioned previously, their plasma levels decrease rather than increase. Also, the plasma enzyme activities are more variable than those of other enzymes. However, these enzymes are useful in assessing neurotoxicity (see Chapter 11). 

Dry chemistry tests are used for the assay of metaboHtes by concentration or by activity in a biological matrix. In general, reactive components are present in amounts in excess of the analyte being deterrnined to make sure that the reactions go to completion quickly. Other enzymes or reagents are used to drive the reactions in the desired direction (25). Glucose and cholesterol are the analytes most commonly measured. 

The oranges were washed, chopped in a meat mincer and homogenised by a Fryma mill. Water (0.6 volumes) were added before the slurry was heat treated by steam injection at 100°C for 2 minutes. The enzyme treatment was carried out for 1 hour at 40°C with 10 lU/g slurry of PME and 25 pg enzyme protein/g slurry of the other enzymes for each of the enzymes. The gelated orange slurry were treated at 85°C for 3 minutes to inactivate the enzymes before the strength of the gel was measured by a SMS TeJrture Analyser TA-XT2 (Stable Micro Systems, XT. RA Dimensions, Operations Manual versions) by compression analysis using a flat cylinder (20 mm dia.) with a speed of 2 mm/s. The force to provide a 20% compression was recorded. 

Establish the validity of using two specific erythrocyte enzyme measurements as sensitive indicators of very low body burdens of lead and other heavy metals... 

Other enzymes have also been immobilized on CNTs for the construction of electrochemical biosensors. Deo et al. [115] have described an amperometric biosensor for organophosphorus (OP) pesticides based on a CNT-modified transducer and OP hydrolase, which is used to measure as low as 0.15 pM paraoxon and 0.8 pM parathion with... 

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

Methods used to demonstrate the existence of membrane phospholipid asymmetry, such as chemical labelling and susceptibility to hydrolysis or modification by phospholipases and other enzymes, are rmsuitable for dynamic studies because the rates of chemical and biochemical reactions are of a different order compared to the transmembrane translocahon of the phospholipids. Indirect methods have therefore been developed to measure the translocation rate which are consequent on the loss of membrane phospholipid asymmetry. Thus time scales appropriate to rates of lipid scrambling under resting conditions or when the forces preserving the asymmetric phospholipid distribution are disturbed can be monitored. Generally the methods rely on detecting the appearance of phosphatidylserine on the surface of cells. Methods of demonstrating Upid translocation in mammalian cells has been the subject of a recent review (Bevers etal., 1999). 

Other stndies have examined the association between the activity of TPMT and other enzymes in the pnrine pathway and AZA toxicity. In one stndy, TPMT, HPRT, 5 -nncleotidase, and pnrine nncleoside phosphorylase activity in the RBCs of 33 RA patients on AZA (dose of approximately 2mg/kg/d) and 66 controls was meas-nred. Compared to patients with normal TPMT activity, 14 RA patients with low (p = 0.004) and 7 patients with intermediate TPMT activity (RR 3.1) developed AZA toxicity(4d). None of the patients were genotyped. Another study measured TPMT activity in 3 RA patients who had experienced AZA-induced hematologic toxicity and 16 RA patients withont AZA toxicity. In this study, 2 patients with AZA-indnced hematologic toxicity were TPMT deficient, one partial and the other complete (50). Patients were not genotyped in either of these studies. 

Several other UV/Vis-based ee assays have been developed, but their general application in directed evolution remains to be demonstrated 66-70). One of them is a well- designed screening system based on enzyme immunoassays (85). The success of the ee assay depends upon the availability of specific antibodies that are easily raised for almost any chiral product of interest. In a given reaction, two antibodies are needed, one that measures the product concentration and the other that measures the amount of one of the enantiomers. About 1000 ee determinations are possible per day, the precision amounting to + 9% (85). 

Lawrence and Sanderson proposed another micro-method for measuring chymosin and other proteolytic enzymes. Measurement of concentration was based on the rate of radial diffusion of the enzyme through a thin layer of caseinate-agar gel. The limit of diffusion was marked by a zone of precipitated casein (Emstrom and Wong 1974). Holmes et al (1977) developed a microdiffusion assay for residual proteolytic enzymes in curd and whey that is more sensitive than the method of Lawrence and Sanderson or the clotting-time assay of Reyes (1971). 

Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur. Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute. Table 1.1 contains such a series of count rates obtained with a scintillation counter on a single radioactive sample. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample (the counting rates, the velocity of the reaction, or protein concentration) and dividing the total by the number of times the measurement was made. The mean is defined by Equation 1.1. 

This method is based on the fact that only esters, but not free carboxylic acids, react with hydroxylamine to form hydroxamic acids, which can be determined colorimetrically as complex with ferric chloride (8). The method—in contrast to most other procedures—measures the concentration of remaining substrate instead of products of hydrolysis. It also requires purified enzymes because of the interference of colored contaminants in the colorimetric measurements. 

All components of the RAS can be found in the brain, heart, vasculature, adipose tissue, gonads, pancreas, placenta, and kidney, among others. Biochemical measurements of ACE activity show that the enzyme is tissue-based. Indeed, <10% of ACE is found circulating in the plasma [4]. The potential importance of the tissue RAS is supported by observations that the beneficial effects of RAS blockers cannot reliably be predicted by measurements of the activity of the circulating RAS. The antihypertensive actions of ACE-inhibitors are better correlated with inhibition of tissue ACE rather than plasma ACE, and hypertensive patients with normal or even low levels of systemic RAS activity can be effectively treated with inhibitors of the RAS. The intrarenal RAS is hypothesized to regulate systemic blood pressure and aspects of renal function such as blood flow and sodium reabsorption. In the brain, the RAS may facilitate neurotransmission and stimu-... 

In doing these calculations, the first goal is to associate the experimentally determined activation parameters for the enzyme catalyzed reaction with a particular reaction mechanism - ideally, to the exclusion of other alternative mechanisms. In order to accomplish this, the calculations employed must first be able to accurately reproduce the experimental free energy of activation (AG ). In the simplest situation, this will only be possible for one type of mechanism in practice, however, there may be several mechanistic pathways with similar barriers (i.e., whose difference is smaller than the error bars on the particular type of calculation). When this is the case, computational predictions of other experimentally measurable quantities - such as KIEs (see Section 2) and changes in rate upon mutation of specific protein residues - may allow for differentiation between mechanisms with similar activation parameters. 

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