It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.
1. A number of mechanistic possibilities exist for P450-catalysed N-dealkylation and have been considered over the years, including C- and N-hydroxylation and sequential electron transfer (SET). With peroxidases the evidence strongly favours SET and free radicals can be detected. Any mechanism must account for lack of incorporation of label from H218O into product by P450s and the high kinetic deuterium isotope effects that are seen in N-dealkylation reactions catalysed by peroxidases but not P450s. 2. Several lines of evidence support a role for SET in P450 amine oxidations, including Hammett analysis, products of dihydropyridine oxidations, and products of mechanism-based inhibition by strained cycloalkylamines. 3. The hypothesis was considered that the P450s act via base catalysis to deprotonate the aminium radical generated by SET, since the pKa has been estimated to be approximately 9. Dihydropyridine aminium radicals have low pKa (< 4) and are generally considered to have considerable kinetic acidity. None of the haemoproteins under consideration (including the peroxidases and haemoglobin) showed high kinetic hydrogen isotope effects for the oxidation of [4-2H]- or [4-3H]-labelled 1,4-dihydropyridines. These results are consonant with the view that P450s catalyse the deprotonation of N,N-dialkylaniline aminium radicals. 4. Since low isotope effects were seen with biomimetic metalloporphyrin models as well as P450s, the deprotonation is attributed to the (FeO)2+ entity, expected to be a strong base, and not the apoprotein. Thus, the FeO moiety of peroxidases is shielded, consistent with evidence by others that SET occurs through the porphyrin edge. Both P450s and peroxidases catalysed the oxidative N-demethylation of aminopyrine and N,N-dimethylaminothioanisole; however, only the peroxidases generated the stable coloured aminium radicals. 5. The rates of N-demethylation of variously para-substituted N,N-dimethylanilines can be used to undertake Hammett or Marcus analysis. The former yields rho = -0.6 and the latter an apparent E1/2 of approximately 1.8 for the formal (FeO)3+ entity of P4502B1. 6. Even in the oxidation of N,N-dialkylanilines, a finite rate of N-oxidation is seen (approximately 0.1% of N-dealkylation). The simplest paradigm has N-oxygenation and N-dealkylation both proceeding from a common aminium radical intermediate.
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