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Selected Metabolic Poisons
organization of
the study Here is a list of poisons that can be used in the study of oxygen consumption
polarography by mitochondria, including sources and considerations for their use.
calibrating
research paper Electron transport inhibitors
Rotenone
Mitochondria theory Antimycin
Cyanide
overview Malonate (succinate dehydrogenase inhibitor)
structure Uncoupling agents
Krebs reactions 2,4-Dinitrophenol (DNP)
electron Carbonyl cyanide p-[triuoromethoxy]-phenyl-hydrazone
transport (FCCP)
the gradient Oligomycin (inhibitor of oxidative phosphorylation)
oxidative
phosphorylation With the exception of malonate and cyanide, these poisons are much more
soluble in ethanol than in water. Adding even a small quantity of ethanol to an
Mitochondria in aqueous medium increases its capacity for oxygen. A Clark electrode detects
vitro such an increase as a temporary rise in oxygen content, followed by the
steady state that would be expected after the addition of the agent. We refer to
this 'blip' on the record as an ethanol artifact.
preparation
fate of
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12/2/2016 Metabolic poisons
Antimycin
Cyanide
Cyanide is an extremely effective reversible inhibitor of cytochrome oxidase.
A concentration of 1 mM KCN is sufcient to inhibit oxygen consumption by
mitochondria from a vertebrate source by >98%. For a nominally 2 ml
chamber, a convenient concentration for the stock solution would be 0.5M (20
l produces a 2.5 mM nal concentration).
dry chemical should be stored under lock and key. As we know from the
Tylenol incidents of a number of years ago, a 500 mg capsule can hold
enough cyanide to kill a person. Because of its volatility, exposure to fumes
from large quantities is hazardous.
Malonate
Malonate (malonic acid) has long been known to inhibit cellular respiration.
Among the key observations made in the 1930s investigations into the nature
of cellular respiration was that the addition of fumarate, malate, or
oxaloacetate to cell preparations resulted in the accumulation of succinate in
the presence of malonate. Malonate is in fact a competitive inhibitor, and
although we treat it as an inhibitor of electron transport it really is an enzyme
inhibitor.
Uncoupling agents
Uncoupling is dened as a condition in which the rate of electron transport
can no longer be regulated by an intact chemiosmotic gradient. The condition
is differentiated from electron transport inhibition by the fact that in the latter
case, bypassing the block can restore the gradient. In uncoupling, the electron
transport system is uninhibited due to complete and irreversible dissipation of
the chemiosmotic gradient.
2,4-Dinitrophenol
DNP is known to have mixed actions, that is, it produces other effects in
addition to uncoupling. DNP gradually inhibits electron transport itself as it is
incorporated into mitochondrial membranes. The effects appear to depend on
concentration of DNP and of mitochondria, and vary from one preparation to
the next.
Back in the 1930s DNP was touted as an effective diet pill. Indeed, the
uncoupling of electron transport from ATP synthesis allows rapid oxidation of
Krebs substrates, promoting the mobilization of carbohydrates and fats, since
regulatory pathways are programmed to maintain concentrations of those
substrates at set levels. Since the energy is lost as heat, biosynthesis is not
promoted, and weight loss is dramatic. However, to quote Efraim Racker (A
New Look at Mechanisms in Bioenergetics, Academic Press, 1976, p. 155),
..."the treatment eliminated not only the fat but also the patients,...This
discouraged physicians for awhile..."
(FCCP)
Oligomycin
Oligomycin, an antibiotic, acts by binding ATP synthase in such a way as to
block the proton channel. That is the mechanism by which oligomycin
inhibits oxidative phosphorylation. Experimentally, oligomycin has no effect
on state IV respiration, that is, it has no direct effect on electron transport or
the chemiosmotic gradient. On the other hand oligomycin prevents state III
respiration completely. To draw the conclusion that an agent is an inhibitor of
ATP synthase (inhibitor of oxidative phosphorylation), the above conditions
must be demonstrated experimentally and unequivocally.
It takes awhile for the effects of oligmycin to show up. Attempts to interrupt
state III respiration by adding oligomycin may fail because of the delay.
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