SH-group reagents as tools in the study of mitochondrial anion transport

Fonyò, Attila

doi:10.1007/bf00743106

Cited by 78 publications

(24 citation statements)

References 84 publications

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“…1). In order to gain an initial insight into the occurrence of carrier-mediated HYP transport, in the same experiment we also examined the ability of mersalyl and phenylsuccinate, used as impermeable inhibitors of certain mitochondrial carriers [15][16][17][18], to affect the rate of fluorescence increase. Both mersalyl (250 laM) and phenylsuccinate (5 mM) significantly decreased (about 55 and 40%, respectively) the rate of intramitochondrial NAD(P)H formation (not shown), thus suggesting that HYP transport is a carrier-mediated process.…”

Section: Spectroscopic Study Of Hydroxyproline Transport In Ratmentioning

confidence: 99%

Carrier‐mediated transport controls hydroxyproline catabolism in heart mitochondria from spontaneously hypertensive rat

Atlante

Seccia²,

Marra

et al. 1996

FEBS Letters

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In this study we have investigated hydroxyproline transport in rat heart mitochondria and, in particular, in heart left ventricle mitochondria isolated from both spontaneously hypertensive and Wistar-Kyoto rats. Hydroxyproline uptake by mitochondria, where its catabolism takes place, occurs via a carrier-mediated process as demonstrated by the occurrence of both saturation kinetics and the inhibition shown by phenylsuccinate and the thiol reagent mersalyl. In any case, hydroxyproline transport was found to limit the rate of mitochondrial hydroxyproline catabolism. A significant change in Vmax and Km values was found in mitochondria from hypertensive/hypertrophied rats in which the Km value decreases and the Vma x value increases with respect to normotensive rats, thus accounting for the increase of hydroxyproline metabolism due to its increased concentration in a hypertrophic/hypertensive state.Key words': Hypertension; Hydroxyproline; Mitochondria; Transport; Collagen major determinant of its architecture, structural integrity and mechanical properties [8].In this paper, we first show HYP carrier-mediated uptake in rat heart mitochondria, after which investigation is made of HYP transport in left ventricle mitochondria in a study carried out with spontaneously hypertensive rats and normotensive Wistar-Kyoto rats, both at 5 and 24 weeks of age. This is worthy of special attention since both the collagen concentration and the total amount of hydroxyproline content in the left ventricle were found to increase in spontaneously hypertensive rats as a result of increased fibroblastic activity in the pathogenesis of cardiac hypertrophy [8]. An increase in HYP translocator activity in hypertrophy/hypertension was shown. In all animals investigated, HYP transport across the mitochondrial membrane was found to limit the HYP oxidation rate. Materials and methods

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Section: Spectroscopic Study Of Hydroxyproline Transport In Ratmentioning

confidence: 99%

Carrier‐mediated transport controls hydroxyproline catabolism in heart mitochondria from spontaneously hypertensive rat

Atlante

Seccia²,

Marra

et al. 1996

FEBS Letters

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“…The final mitochondrial pellet was resuspended in the medium to obtain 30 -40 mg protein/ml. Control experiments showed that the phosphate carrier was completely blocked in mersalyl-treated rat kidney mitochondria, as monitored according to [13], and that any intramitochondrial transaminase was strongly inhibited.…”

Section: Treatment With Mersalyl and Aminooxyacetatementioning

confidence: 99%

Ornithine/phosphate antiport in rat kidney mitochondria

Passarella¹,

Atlante²,

Quagliariello³

1990

European Journal of Biochemistry

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['4C]Ornithine uptake by rat kidney mitochondria has been investigated according to the stop inhibitor method by using praseodimium chloride as an inhibitor. The existence of an ornithine/Pi exchange was found occurring with 1 : 1 stoichiometry. Both uptake and efflux follow first-order kinetics with a k of 2.4 min-'. Uptake increases with increasing pH. The activation energy for the process is 58.6 kJ/mol and Qlo is 2.6. Ornithine/P, exchange is electrical and energy-dependent, as suggested by the sensitivity of the process to the ionophores valinomycin and nigericin. Measurements both of proton movement across the mitochondrial membrane and of membrane potential strongly suggest that ornithine uptake into mitochondria is driven by the electrochemical proton gradient via the dependent ornithine/Pi translocator and ApH-dependent Pi carrier.Mitochondria can only function if there is a continual interchange of metabolites and end products with the cell cytosol; thus about 12 different transporters have been identified in mammalian mitochondria [l] to allow for movement of substrates from cytosol to mitochondria and vice versa. However further investigation seems necessary both to ascertain the occurrence of such translocators in mitochondria from different sources and to identify the existence of special carriers involved in specific metabolism. In particular, in kidney in addition to glutamate/aspartate [2], dicarboxylate [3], glutamine [4, 51, oxoglutarate [6], fumarate [7] and glutamate/ OH- [8], we have shown the existence of malate/oxaloacetate and pyruvate/oxaloacetate translocators [9]. Recently the existence in kidney mitochondria of an ornithine/Pi translocator has been shown by fluorimetric and photometric measurements [lo]. To gain further insight into ornithine uptake by rat kidney mitochondria, an isotopic study appears worthwhile. Thus this paper deals with experiments in which [14C]ornithine uptake by rat kidney mitochondria has been measured according to the stop inhibitor method and certain aspects of the process investigated in detail. Ornithine/Pi exchange has been proved to occur electrically with a 1 : 1 stoichiometry. MATERIALS AND METHODS MaterialsAminooxyacetic, L-glutamic, L-malic and methylglutamic and other amino acids, L-ornithine, mersalyl, EGTA, Triton X-100, rotenone, nigericin, valinomycin, carbonylcyanide ptrifluoromethoxyphenylhydrazone (FCCP), safranine 0 and metal chlorides were obtained from Sigma (St Louis, USA);

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“…5. pCMBS is a membrane impermeable reagent whose reaction with thiols can be reversed by addition of excess dithiothreitol or mercaptoethanol [4]. MalNEt on the other hand is membrane permeable and reacts irreversibly with SH groups.…”

Section: The Involvement Of Afunctional Thiol Group In the Proline Cmentioning

confidence: 99%

The location of redox‐sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

Poolman

Konings

Robillard

1983

European Journal of Biochemistry

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Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane.Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the A f i H +. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide.Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a A,iH+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group.Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a A&+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.Sulphydryl groups play an important role in the function of many bacterial, chloroplast and mitochondria1 transport and energy transducing systems [I -61. Many of these membranebound proteins contain dithiol groups which could play a role in transport and other membrane related processes [7-141.Recently we presented evidence that dithiol-disulphide interchanges effect the activity of three different transport systems : the Escherichiu coli phosphoenolpyruvate-dependent glucose transport system [15] and the E. coli proton-symport transport systems for proline and lactose [16]. The reduced dithiol form has a low K,,, for solute and the oxidized disulphide has a high K,. The changes between the oxidized and reduced states can be generated either by artificially changing the redox potential with oxidizing and reducing agents or by establishing a AilH+ across the membrane. On the basis ofthese observations and of similar reports on other systems we proposed that dithiol-disulphide interchange could play a general role in transport and energy-transducing processes [I 71. One element of this hypothesis is that dithiols and disulphides are located ...

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SH-group reagents as tools in the study of mitochondrial anion transport

Cited by 78 publications

References 84 publications

Carrier‐mediated transport controls hydroxyproline catabolism in heart mitochondria from spontaneously hypertensive rat

Carrier‐mediated transport controls hydroxyproline catabolism in heart mitochondria from spontaneously hypertensive rat

Ornithine/phosphate antiport in rat kidney mitochondria

The location of redox‐sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

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