Problems with enzyme studies

Finally, perhaps the greatest problem with enzymatic studies of Pol and Revlp concerns the properties and structure of the native enzymes. As in the case of P0I77, for which a considerable enhancement of insertion efficiency was observed following association with PCNA and the presence of RPA (Haracska et al, 2001b,d), the properties of Pol and Revlp may also be enhanced by such factors. In particular, the apparent inefficiency of Pol for insertion opposite lesions in vitro may reflect their absence. A variety of evidence suggests that Pol and Revlp are associated with one another in a multiprotein complex of some kind, possibly because Revlp acts as a structural Unk between Pol and PCNA (see Section V). Identifying and investigating the enzymatic properties of such a complex is likely to be needed for a fuller characterization of these proteins. 

Toxicological studies have demonstrated that there are no important problems with fluconazole. Therapeutic doses of fluconazole may cause enzyme induction in the Hver. This suggests that interactions with other dmgs cannot be excluded. The side effects are similar to those of itraconazole and include nausea, headache, and vertigo. Occasionally, increased Hver enzymes may be noted. Like itraconazole, fluconazole is contraindicated during pregnancy. 

Similar to lipids the oxidation of proteins has already been studied for more than 20 years. Before discussing the data on protein oxidation, it should be mentioned that many associated questions were already considered in previous chapters. For example, the oxidation of lipoproteins, which is closely connected with the problems of nonenzymatic lipid peroxidation was discussed in Chapter 25. Many questions on the interaction of superoxide and nitric oxide with enzymes including the inhibition of enzymatic activities of prooxidant and antioxidant enzymes are considered in Chapters 22 and 30. Therefore, the findings reported in those chapters should be taken into account for considering the data presented in this chapter. 

Monoamine Oxidase Inhibitors (MAOIs). The MAOls work in a unique fashion by blocking the activity of an enzyme that degrades each of three key brain transmitters norepinephrine, dopamine, and serotonin. These widespread effects on several brain transmitter systems make the MAOls a potentially very effective class of medications for a variety of disorders. A few small studies have evaluated the usefulness of the MAOls in the treatment of BPD and found them moderately helpful for the impulsivity associated with this illness. Unfortunately, the requirements for strict dietary restrictions due to a risk of hypertensive crisis severely limit the usefulness of MAOls in the treatment of BPD. These restrictions are a particular concern when treating patients who have problems with impulsivity and are therefore likely to have difficulty maintaining the dietary regimen. For this reason, although they may theoretically be helpful, MAOls should only be used to treat BPD after other more easily tolerated medications have been tried and have failed. In the near future, so-called reversible MAOls that appear to avoid the need for diet restrictions may become available. If so, this will allow us to reconsider their use in the treatment of BPD. For more information regarding the use of MAOls, please refer to Chapter 3. 

A problem with toxicity produced by an extension of the pharmacology of a compound, is that the conventional use of no-effect doses based on pre-clinical animal studies may not apply. Moreover pre-clinical studies may be complicated by the often understated ranges of response seen across species due to species differences in the receptors, enzymes and ion channels that comprise drug targets. Table 8.1 lists some of these known variations and the consequences range from an exaggerated response, to an absence of a response. 

While the batch process is the dominant one in current use, researchers and companies have attempted to create continuous bioreactor systems. Lopez et al. immobilized Candida rugosa in polymethacrylamide hydrazide beads and polyurethane foam 3 with the intent to achieve the continuous production of lipase enzymes. Despite flow problems with the polyurethane foam, it showed high lipolytic activity. Biomass buildup was problematic. Feijoo et al. immobilized Phanerochaete chry-sosporium on polyurethane foam in packed bed bioreactors under near-plug flow conditions. Continuous lignin peroxidase production was accomplished, the rate of which was studied as a function of recycle ratio. 

Instability of the mutant AChE can be a problem with up to 50% of its activity in solution being lost in 10 days. This led to a study in which the enzyme was immobilised in porous silica (pore size 10 nm) or porous carbon (<70nm) beads [36]. The AChE is known to be approximately 6nm in size and therefore it is thought that entrapment within the pores could well inhibit unfolding of the enzyme, so enhancing its stability. 

Quantum chemical methods aim to treat the fundamental quantum mechanics of electronic structure, and so can be used to model chemical reactions. Such quantum chemical methods are more flexible and more generally applicable than molecular mechanics methods, and so are often preferable and can be easier to apply. The major problem with electronic structure calculations on enzymes is presented by the very large computational resources required, which significantly limits the size of the system that can be treated. To overcome this problem, small models of enzyme active sites can be studied in isolation (and perhaps with an approximate model of solvation). Alternatively, a quantum chemical treatment of the enzyme active site can be combined with a molecular mechanics description of the protein and solvent environment the QM/MM approach. Both will be described below. 

The diastereomers of Mg-ATP in Fig. 2 exemplify in a limited way the stereochemical problem in metal nucleotides. The two isomers shown are in rapid exchange equilibrium, however, so it is not possible to separate them and study their individual interactions with enzymes. The problem is further complicated by the fact that these diastereomers represent the stereochemical possibilities in only one of the coordination isomers. Others are possible, including the a-, / - and y-monodentate isomers and a,/ ,y-tridentate isomers, most of which exist as two or more diastereoi-somers. All of the coordination isomers and their diastereoisomers of Mg-ATP are in rapid exchange equilibrium. 

Partial proteolysis has been used by several researchers to improve functional properties, i.e. foaming, solubility of proteins (7,8,9). The significant problems associated with enzyme hydrolysis of proteins are excessive hydrolysis occurring under batch conditions, the generation of bitter flavors during hydrolysis and the cost of enzymes. Extensive information on factors affecting proteolysis of proteins and the problem of bitterness has been reviewed by Fujimaki et al. (7) in conjunction with studies of the plastein reaction. 

While most of the problems in the assay of an activity purified by HPLC are expected and typical of chromatographic work with enzymes, the introduction of this technique into the purification scheme may lead to problems if the fractions obtained from the HPLC purification step are to be measured for enzymatic activity. For example, the salt in each fraction may inhibit any enzymatic activities it contains. Moreover, when ion-exchange HPLC is used the salt concentration will vary in the fractions. Thus it is prudent to study the effects of salt, at the concentration used for elution, on enzyme activity before the chromatography. If the salt is found to be detrimental, it will have to be eliminated or at least reduced in concentration before the chromatography. Removing the salt by dialysis may not be the appropriate way to proceed, however, since the inactivation of enzyme activities is not always reversible. 

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