David Njus

Vitamins C and E donate single hydrogen atoms in vivo

The antioxidant vitamins, C and E, eliminate cytotoxic free radicals by redox cycling. Energetic and kinetic considerations suggest that cycling of vitamin C and vitamin E between their reduced and free radical forms occurs via the transfer of single hydrogen atoms rather than via separate electron transfer and protonation reactions. This may enable these vitamins to reduce many of the damaging free radicals commonly encountered by biological systems while minimizing the reduction of molecular oxygen to superoxide.

Proton-Linked Transport in Chromaffin Granules1

Publisher Summary This chapter focuses on proton-linked transport in chromaffin granules. The function of the chromaffin granule is to store catecholamine in high concentration and, upon stimulation of the chromaffin cell, to deliver the catecholamine into the extracellular space by exocytosis. Chromaffin granules isolated from the adrenal medulla are a mixture of two populations, one containing epinephrine and the other containing norepinephrine. The chromaffin-granule adenosine triphosphatase hydrolyzes ATP on the external face of the membrane and translocates protons into the granule. The proton-linked transport system provides a good mechanism for achieving high concentration gradients without wasting energy in pumping catecholamines back and forth across the membrane. Because the catecholamine gradients are essentially in equilibrium with the pH gradient and membrane potential, catecholamines may exchange across the membrane with no energy consumption. Although norepinephrine will tend to stay in the granules, a continuous flux of norepinephrine into the cytoplasm will occur as long as norepinephrine in the cytoplasm is efficiently converted to epinephrine. Studies of catecholamine metabolism in chromaffin granules have benefited immeasurably from techniques and concepts of bioenergetics

Mechanism of Ascorbic Acid Regeneration Mediated by Cytochrome b561

In summary, ascorbic acid serves as a one-electron donor for dopamine beta-hydroxylase in chromaffin vesicles and probably for peptide amidating monooxygenase in neurohypophyseal secretory vesicles. It appears that the semidehydroascorbate that is produced is reduced by cytochrome b561 to regenerate intravesicular ascorbate. Cytochrome b561, a transmembrane protein, is reduced in turn by an extravesicular electron donor, probably cytosolic ascorbic acid. It will be interesting to see whether other ascorbate-requiring enzymes in other organelles use a similar ascorbate-regenerating system to provide an intravesicular supply of reducing equivalents.

Oxidation of 4-methylcatechol: implications for the oxidation of catecholamines.

At alkaline pH, 4-methylcatechol oxidizes more rapidly than the related catecholamines: dopamine, norepinephrine, and epinephrine. This oxidation is not inhibited by superoxide dismutase or catalase, indicating that O2 itself is the oxidant, but the reduction potential of O2/O2-* is too low for it to oxidize 4-methylcatechol directly. Instead, O2 oxidizes the 4-methylcatechol semiquinone, which is formed by comproportionation of 4-methylcatechol and its o-quinone. Aniline reacts very quickly with the o-quinone and thus stops the comproportionation reaction that oxidizes 4-methylcatechol to the semiquinone. Oxidation of 4-methylcatechol then requires superoxide, and in the presence of aniline, oxidation of 4-methylcatechol by O2 is inhibited by superoxide dismutase. When catecholamines oxidize, the side chain amine inserts into the catechol o-quinone, forming a bicyclic compound. By eliminating the quinone, this ring closure prevents comproportionation and the consequent oxidation of catecholamines by O2. It also prevents reaction of the quinone with other compounds and the formation of potentially toxic products.

The epinephrine assay for superoxide: Why dopamine does not work

Superoxide oxidizes epinephrine to a semiquinone, initiating a series of reactions leading to the colored product adrenochrome. This popular assay for superoxide is more sensitive at higher pH, and it does not work if dopamine is used instead of epinephrine. A kinetic analysis shows that these effects can be explained by competing reactions that lower the yield of the observed product. The catecholamine quinone may cyclize to form the absorbing product, or it may be reduced back to the semiquinone by superoxide. For epinephrine, the quinone cyclizes quickly and adrenochrome formation dominates, but for dopamine, the quinone cyclizes slowly and the back reaction prevails. The yield of adrenochrome increases if the epinephrine semiquinone reacts with O(2) to form more superoxide, but this reaction competes with disproportionation of the semiquinone. Because disproportionation slows as pH increases, both superoxide formation and the yield of adrenochrome increase at higher pH.

Concerted proton-electron transfer between ascorbic acid and cytochrome b561.

Ascorbic acid is an essential reductant in biology but its reducing power is paradoxical. At physiological pH the predominant form of ascorbate (the monoanion) is a poor electron donor because it oxidizes to the energetically unfavorable neutral free radical. The ascorbate dianion forms the relatively stable semidehydroascorbate radical anion and is a powerful electron donor but its concentration at neutral pH is insufficient to produce the reaction rates observed. For example, ascorbate rapidly reduces cytochrome b561 from adrenal medullary chromaffin vesicles. This fast reaction rate may be rationalized by a mechanism involving concerted proton-electron transfer rather than electron transfer alone. This would permit reduction of the cytochrome by the abundant ascorbate monoanion but would circumvent formation of unfavorable intermediates. This may be a general mechanism of biological ascorbic acid utilization: enzymes using ascorbic acid may react with the ascorbate monoanion via concerted proton-electron transfer.

Inhibition of norepinephrine transport and reserpine binding by reserpine derivatives

Abstract: Reserpine, a competitive inhibitor of catecholamine transport into adrenal medullary chromaffin vesicles, consists of a trimethoxybenzoyl group esterified to an alkaloid ring system. Reserpine inhibits norepinephrine transport with a Ki of ∼ 1 nM and binds to chromaffin‐vesicle membranes with a KD of about the same value. Methyl reserpate and reserpinediol, derivatives that incorporate the alkaloid ring system, also competitively inhibit norepinephrine transport into chromaffin vesicles with Ki values of 38 ± 10 nM and 440 ± 240 nM, respectively. Similar concentrations inhibit [3H]reserpine binding to chromaffin‐vesicle membranes. 3,4,5‐Trimethoxybenzyl alcohol and 3,4,5‐trimethoxybenzoic acid, derivatives of the other part of the reserpine molecule, do not inhibit either norepinephrine transport or [3H]reserpine binding at concentrations up to 100 μM. Moreover, trimethoxybenzyl alcohol does not potentiate the inhibitory action of methyl reserpate. Therefore, the amine binding site of the catecholamine transporter appears to bind the alkaloid ring system of reserpine rather than the trimethoxybenzoyl moiety. The more potent inhibitors are more hydrophobic compounds, suggesting that the reserpine binding site is hydrophobic.

The chromaffin vesicle and the energetics of storage organelles.

In the chromaffin vesicle, energy for amine transport is provided by a proton-translocating adenosine triphosphatase. The ATPase pumps protons into the vesicle; a pair of protons is then exchanged for each catecholamine taken up. In terms of transport, the adrenal chromaffin vesicle is an important model for non-mitochondrial organelles of all types. These organelles include adrenergic synaptic vesicles, other secretory and neurotransmitter storage vesicles, and lysosomes and other intracellular organelles as well. The membranes of these organelles all possess an ATPase that pumps H+ ions into the vesicles. This proton pump presumably drives the transport of ions and molecules into the organelle and powers other energy-requiring functions of the membrane.

Electron transfer in chromaffin-vesicle ghosts containing peroxidase

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.

A spin-label study of plasma membranes of adrenal chromaffin cells

Chromaffin-cell membranes were labeled with two nitroxide spin labels, one probing the interior of the membrane and one probing the interfacial region. Both spin labels indicate that the membrane undergoes a phase transition at about 26 degrees C. An Arrhenius plot of acetylcholinesterase activity exhibits a discontinuity at 26 degrees C, consistent with the existence of a phase transition at that temperature. Acetylcholine, which stimulates chromaffin cells to secrete catecholamines, and hexamethonium, a cholinergic blocker, do not affect the rotational correlation times of the spin labels. These results argue that cholinergic stimulation does not affect the fluidity of the chromaffin-cell membrane.