Residual enzyme activity

Experiment 3. Estimation of residual enzyme activity in the biotest To calculate the residual activity of AChE-biotest after AChE inhibitors action on the biotest, three measurements will be done (i) measurement Dn of the biotests after incubation with substrate, (ii) measurement Dw/s after incubation without substrate and (iii) measurement Ding after incubation with substrate and inhibitor. The amount of residual activity (A) will be calculated in percentage with the following formula ... 

Presently available methods to diagnose and biomonitor exposure to anticholinesterases, e.g., nerve agents, rely mostly on measurement of residual enzyme activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in blood. More specific methods involve analysis of the intact poison or its degradation products in blood and/or urine. These approaches have serious drawbacks. Measurement of cholinesterase inhibition in blood does not identify the anticholinesterase and does not provide reliable evidence for exposure at inhibition levels less than 20 %. The intact poison and its degradation products can only be measured shortly after exposure. Moreover, the degradation products of pesticides may enter the body as such upon ingestion of food products containing these products. 

Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and >galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and -galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and -galactosidase, respectively, were preincubatcd at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

Treatment for these autosomal recessive conditions is to provide a Met-restricted diet with vitamin supplementation to enhance any residual enzyme activity that may be available and with Cys supplementation to make up for the deficiency in its synthesis. 

Type Il/Pompe usually undetectable, or very low level of enzyme activity in infantile form residual enzyme activity in late-onset. Infant hypotonia, muscle weakness, cardiac enlargement and failure, fatal early... 

The conditions to be diagnosed are frequently heterogeneous in their biochemical expression as a result of varying residual enzyme activity and other confounding genetic and environmental factors. 

If necessary, the size of peptides generated during the experiment can be evaluated using a 10% to 18% gradient SDS-polyacrylamide gel (Laemmli, 1970 unitbs.i). It is very important to be sure that there is no residual enzymatic activity when the desired DH has been reached. To guarantee no residual enzyme activity, a useful technique is substrate SDS-PAGE (Garcfa-Carreno, 1993). 

Fig. 29.7. Effect on the residual enzyme activity of methanol and acetone at different concentration (0-10%) in phosphate buffer. Redrawn with permission from Ref. [49].

Characterization of a mechanism-based inhibitor may involve the estimation of the constants described in section IV, namely, /qnacI, Kh /ccat, and r. The most common approach has been to incubate inhibitor, enzyme, and cofactors together and to determine the decline in enzyme activity with time (26). In practice, this approach often employs the measurement of residual enzyme activity in a subsequent incubation with a specific substrate under conditions that limit further inactivation and competitive inhibition by the inactivator, usually by an appropriate dilution (10-fold or greater) of the original incubate (5). 

Under conditions where it is not possible to approximate the steady state, i.e., constant inactivator concentration, it is possible to estimate /clnact and Kt, if the inactivator concentration and residual enzyme activity are quantified simultaneously. If a fixed quantity of enzyme and inactivator are combined under... 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of... 

Canine MPS I was discovered in a Plott hound that presented with corneal clouding [10]. Studies by Shull and Neufeld showed that the dogs were deficient in a-l-iduronidase [11], Being null, MPS I dogs are genetically similar to the most severe form of MPS I in humans, but clinically they more closely resemble moderately affected patients. These animals provide a valuable bio-chemical/clinical model for MPS I disease. Since they have no confounding residual enzyme activity, they accumulate GAGs in relevant tissues, and their clinical phenotype closely resembles the human disease. 

In general, null alleles are associated with the classic early-onset phenotype, whereas missense mutations which lead to defective proteins that exhibit residual enzyme activity lead to attenuated phenotypes (Froissart et al., 2002). However, studies of genotype-phenotype correlation have revealed a lack of perfect concordance, which suggests other factors may be involved that influence disease outcome (Froissart et al., 2002). At present, the putative factors that modify LSD-phenotypes among patients with identical genotypes remain obscure. 

Infants with benign hyperphenylalaninemia are occasionally identified because of a moderately elevated blood concentration of phenylalanine. These patients have a partial deficiency of phenylalanine hydroxylase with residual enzyme activity up to 35% of normal subjects. Although detected by neonatal screening, they remain healthy without dietary treatment once the possibility of an underlying cofactor deficiency has been ruled out. 

Although ornithine transcarbamylase is fairly widely distributed in the tissues, only the enzyme levels in the liver in hyperammonemia have been adequately reported (Table 7), except that in one affected infant and the mother of two others, the small intestinal mucosa has also been examined (L8). In a series of 8 children, 7 females and 1 male, the liver ornithine transcarbamylase activity was 10% or less of the mean normal value, with the exception of the male, in whom the residual enzyme activity was as much as 25% of the mean normal. In 6 cases in whom the activity was also determined at pH 8.3, the reduction was much more variable, the activity varying from 11% to 40% of the mean normal an exception is the case mentioned previously, in whom the activity was actually within the normal range at that pH although slightly lower than the mean normal. These results together with the patient reported by Hopkins et al. (H5) proved conclusively that hyperammonemia is due to a gross deficiency of ornithine transcarbamylase activity. One other case, reported by Corbeel et al. (C12, C13), showed a somewhat... 

The IC50 was calculated from plots of residual enzyme activity against inhibitor concentration, using Origin Scientific plotting software (Northampton, MA, USA). 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of denaturation occurs with the immobilized enzyme, which may involve a two-phase process. Immobilization offers more resistance to denaturation at the higher temperature of 60°C where the second phase is prolonged by a factor of three [60]. Differential scanning calorimetry experiments showed that bromelain is an exceptional protease among the cysteine proteases, illustrated by the fact that its thermal denaturation is consistent with an irreversible two-state model [61]. Also, the far UV circular dichroism spectrum of bromelain differs from those of papain and chymopapain and therefore represents a third spectral class within the cysteine proteinase family [62],... 

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