[26] Direct methods for measuring metabolite transport and distribution in mitochondria
Methods in enzymology on CD-ROM/Methods in enzymologyPublished 1 January 1979
Ferdinando Palmieri, Martin Klingenberg
Citations190
SJR quartileQ4
SJR score0.13
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TL;DR
This chapter describes the direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria, and the measurements of metabolite distribution are made after the removal of mitochondria from the incubation medium.
Abstract
This chapter describes the direct methods for studying mitochondrial transport, which are necessarily linked to the techniques for separating the mitochondria. Since the rates of transport reactions are higher in mitochondria than in whole cells and bacteria, special emphasis will be devoted to the techniques developed for measuring kinetics. It is possible to study (1) the efflux of endogenous substrates or of substrates from previously loaded mitochondria, (2) the uptake of added metabolites, and (3) the counterexchange by following the in and out movements of the exchanging metabolites. The steady state distribution and the kinetic of transport between the two spaces are also examined. The measurements of metabolite distribution are made after the separation of mitochondria from the incubation medium. It is emphasized that the amount of metabolite within the mitochondria is relatively small, since the volume of the suspending medium is orders of magnitude larger than that of the intramitochondrial space.
Keywords
MedicineBiochemistry, Genetics and Molecular Biology
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The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed, and alpha-cyano-4-hydroxycinnamate was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion), which allowed measurements of pyruVate transport at higher temperatures to be made.
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It is brought out that the light-scattering method fails to distinguish between changes in sucrose space and in Sucrose-inaccessible space, which in the present experiments could be altered respectively by phosphate (in high K(+) solution) and by cation uptake induced by antibiotic.
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The rate of uptake in mitochondria as catalyzed by the dicarboxylate carrier of dicARboxylates and, in the presence of N-ethylmaleimide, of inorganic phosphate, has been analyzed by using the inhibitor stop method, and it is concluded that the carrier has two separate binding sites, one specific for Pi and the other specific for the dICar boxylates.
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Investigation in rat heart mitochondria of interactions of glutamate metabolism with transport steps across the mitochondrial membrane and enzyme steps of the citric acid cycle found regulation is complementary to control of oxalacetate availability by the intramitochondrial NADH:NAD ratio which also regulates α-ketoglutarate production via both citrate synthase and aspartate aminotransferase.
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84 Citations1970B. H. Robinson, G. R. Williams
The exchange of the labelled material with P i was more sensitive to inhibition by 2- n -butylmalonate than the exchange with dicarboxylate anions, and the rates of exchange and the concentration dependence were determined using the ‘inhibitor stop’ technique.
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63 Citations1973Norah M. Bradford, J D McGivan
The kinetics of glutamate transport into mitochondria were determined by using Bromocresol Purple to terminate the transport process and suggest that glutamate deamination may be controlled by the rate of glutamate Transport.
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It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).
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56 Citations1969Ferdinando Palmieri, E Quagliariello
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Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane and it is proposed that for reasons of control the cell will require membrane monoccarboxylate transporters no less than di- or tri-carboxyate carriers.
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51 Citations1974Ferdinando Palmieri, Salvatore Passarella +2 more
It is concluded that SH reagents bind at or near the dicarboxylate specific binding site and distant from the Pi binding site, resulting in inhibitors that prevent dicarsalylate binding directly and decrease the affinity for Pi by an indirect conformational change.
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It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier.
Biochemical JournalA kinetic study of sulphate transport in rat liver mitochondria
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It is considered that sulphate and malonate probably bind to the same locus on the carrier, whereas phosphate occupies a different site, and the possible implications of this conclusion are discussed.
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11 Citations1971Ferdinando Palmieri, Giuseppe Genchi +1 more
The study of the regulation of anion distribution across the mitochondrial membrane has been extended to other physiologically important substrates, e.g. glutamate, pyruvate and aspartate, and as partate was found to be the only anion whose distribution seems to be dependent on membrane potential but not on ΔpH.
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It is concluded that the uncoupling effect is not related to the interaction of uncouplers with substrate carriers or to their entry on these transport systems, and the inhibition of substrate uptake at equilibrium can be accounted for only by the collapse of the ΔpH.