Enzyme inactivation in vivo

Whether a toxin is naturally reactive to biological macromolecules or receptors, or requires metabolic activation to produce such a species, e.g. the enzyme-mediated transformation of tremorine (9) to the active parasympathomimetic agent oxotremorine (10), it will usually be subject to chemical or enzymic inactivation in vivo. Interruption of the latter process via appropriate substitution may thus lead to an increase in biological activity or toxicity over that of the parent compound. Perhaps the most striking example of this is provided by the extreme metabolic stability and toxicity of TCDD and the nontoxicity of its de- chloro analogue dibenzodioxin (Table 3). 

Direct evidence that irreversible inhibition is the principle mechanism underlying in vivo drug-drug interactions (DDIs) is often lacking because of the requirement for either direct tissue sampling to reveal inactivated enzyme or in vivo inhibition of activity after drug is essentially eliminated from the body. Nevertheless the steady-state plasma concentrations of several clinically important CYP inhibitors are well below the in vitro estimated competitive inhibition constant, Kv This suggests that competitive inhibition is unlikely to occur in vivo, yet these compounds inhibit CYP activity in a time and concentration-dependant manner when cDNA-expressed CYPs or HLMs are used as an enzyme... 

Insulin is inactivated in vivo by (a) an immunochemical system in the blood of insulin-treated patients, ib) reduction of disulfide bonds (probably by glutathione), and (c) insu-linase (a proteolytic enzyme) that occurs in liver. Pepsin and chymotrypsin hydrolyze some peptide bonds that lead to... 

Lack of Inactivation by Energy-yielding Cellular Metabolism. We first examined whether the dependence of synthetase Inactivation on oxygen reflects a requirement for aerobic energy-yielding metabolism. Some enzymes are Inactivated In vivo In the presence of... 

The physiological significance of in vitro reversible inactivation, affecting the oxidation-reduction status of the enzyme, is not readily apparent. In those instances where the enzyme is rapidly inactivated in vivo, such mechanisms may be affecting regulation, however, in other instances where curtailment ot nitrate utilization occurs without a rapid decline in extractable activity, other systems must be involved. 

Vennesland s group has concluded that the inactivation in vivo after the cells have been treated with ammonia involves the formation of a firmly bound complex of reduced enzyme and cyanide. They have speculated that the product of ammonium assimilation which inhibits nitrate reductase is cyanide. In this respect the COj requirement for inactivation by ammonia in the light could be relevant. On the other hand, COg could potentiate the stimulating effect of ammonia on the photosynthetic non-cyclic electron flow.< If this interpretation would be correct, the activation of the ADP-dependent pyruvate kinase reaction by ammonia discovered by Bassham in Chlorella might be better explained by an increase in the cellular ADP level induced by the uncoupling effect of ammonia on photophosphorylation than by a direct activation of the kinase by the ammonium cation, as it has been postulated. 

Litde is known about metaboHc inactivation of ( -endorphin and the dynorphins. NEP, and to a lesser extent APN, are only weaMy active against P-endorphin (183). Enzymes are known which degrade P-endorphin in vitro under nonphysiological conditions (202) or which inactivate P-endorphin by N-acetjlation (203). Alack of specific degradative enzymes for these peptides may account for their relatively long half-life in vivo though this has not been definitively estabUshed. 

Calmodulin, a calcium binding protein, is involved in Ca2+-dependent regulation of several synaptic functions of the brain synthesis, uptake and release of neurotransmitters, protein phosphorylation and Ca+2 transport. It reacts with TET, TMT and TBT which then inactivates enzymes like Ca+2-ATPase and phosphodiesterase. In vitro studies indicated TBT was greater at inhibiting calmodulin activity than TET and TMT, whereas in vivo the order was TET > TMT > TBT. This may be due to the greater detoxification of TBT (66%) in the liver before moving to other organs30,31. 

An interesting dinically useful prodrug is 5-fluorouracil, which is converted in vivo to 5-fluoro-2 -deoxyuridine 5 -monophosphate, a potent irreversible inactivator of thymidylate synthase It is sometimes charaderized as a dead end inactivator rather than a suicide substrate since no electrophile is unmasked during attempted catalytic turnover. Rathei since a fluorine atom replaces the proton found on the normal substrate enzyme-catalyzed deprotonation at the 5 -position of uracil cannot occur. The enzyme-inactivator covalent addud (analogous to the normal enzyme-substrate covalent intermediate) therefore cannot break down and has reached a dead end (R. R. Rando, Mechanism-Based Enzyme Inadivators , Pharm. Rev. 1984,36,111-142). 

To obtain tissue preparations whose constituents were maintained as closely as possible to their state in vivo, the material had to be fixed, i.e. the enzymes inactivated so that cell structures were instantaneously preserved, an almost unattainable ideal. Formalin was the favored fixative, but others (e.g. picric acid), were also employed. Different methods of fixation caused sections to have different appearances. Further artifacts were introduced because of the need to dehydrate the preparations so that they could be stained by dyes, many of which were lipid-soluble organic molecules. Paraffin wax was used to impregnate the fixed, dehydrated material. The block of tissue was then sectioned, originally by hand with a cut-throat razor, and later by a mechanical microtome. The sections were stained and mounted in balsam for examination. Hematoxylin (basophilic) and eosin (acidophilic) (H and E staining) were the commonest stains, giving blue nuclei and pink cytoplasm. Eosinophils in the blood were recognized in this way. 

The hydroxylamine or vinylglycine analogues are potent inhibitors of PLP-dependent enzymes in vivo and in vitro. The hydroxylamine analogues inactivate the enzyme forming stable oximes with PLP. 

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