Energy-dependent uptake of arsenite by rat liver mitochondria.
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ABSTRACT:
SUBMITTER: Harris EJ
PROVIDER: S-EPMC1184124 | biostudies-other | 1977 Oct
REPOSITORIES: biostudies-other
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Project description:Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1delta sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.
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Project description:The rate and extent of Cu2+ uptake by rat liver mitochondria was measured under various conditions. 1. The uptake is both greater and faster without an energy supply. 2. The uptake, when occuring in ionic media, has a biphasic character, that is it always slows down after an initial burst, and then re-accelerates. 3. Uptake of Cu2+ in the presence of energy initiates K+ uptake from K+-containing media with accompanying swelling and respiratory stimulation. Depending on the amounts of Cu2+ added and the K+ concentration, an inhibition of respiration later ensues. 4. Chelation of the Cu2+ by substrates (notably glutamate) decreases the effects. 5. Prior exposure to Cu2+ decreases or prevents energy-dependent Ca2+ uptake.
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Project description:1. To gain insight into a putative role for mitochondria in silicon metabolism, mitochondrial uptake (by which it is meant the removal from the medium) of silicic acid [Si(OH)4] was studied under conditions minimizing SI(OH)4 polymerization. 2. Measurements of mitochondrial respiration and swelling indicated indirectly a significant uptake of Si(OH)4 as a weak acid, but this was not confirmed when 31Si(OH)4 was used as a tracer. 31Si(OH)4 occupied a mitochondrial volume similar to that of 3H2O and was relatively unaffected by mitochondrial energy status and by the pH gradient across the mitochondrial inner membrane. 3. Uptake was directly proportional to Si(OH)4 concentration in the range 0-3 mM. 4. The uptake consisted of two components: under all conditions examined, the greater quantity, amounting to 1-2nmol of Si(OH)4/mg of mitochondrial protein, was bound, a major portion of it external to the inner membrane, with the lesser quantity free within the matrix space. 5. Equilibration of 31Si(OH)4 between medium and matrix was a slow process, having a half-time of approx. 10 min at 22 degrees C. 6. Mersalyl and N-ethylmaleimide inhibited the uptake by preferentially lowering the amount of Si(OH)4 bound. Their action was somewhat variable, depending on the precise nature of the suspending medium, and suggesting that the bound material may represent polymerized forms of Si(OH)4. 7. It is concluded that Si(OH)4 may penetrate the mitochondrial inner membrane by a simple diffusion mechanism.
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Project description:Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.
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Project description:In rat liver mitochondria suspended in KCl medium and containing a low concentration of a K(+)-specific cationophore (valinomycin or Triton X-100), oligomycin was shown to induce uncoupling of oxidative phosphorylation, stimulation of adenosine triphosphatase activity, release of the respiratory control, decrease of energy-dependent changes in the fluorescence of the dye 8-anilino-1-naphthalenesulphonic acid and rapid swelling of mitochondria. Oligomycin caused none of the above effects when Br(-) or NO(3) (-) was substituted for Cl(-) as the major anionic species or when Na(+) replaced the K(+). The same concentration of oligomycin that caused uncoupling and swelling slightly improved energy-conserving reactions when the cationophores were omitted. In the presence of KSCN, valinomycin or Triton X-100 by itself caused uncoupling and swelling which was not further enhanced by oligomycin. On the basis of the above results it is suggested that the energy dissipation resulting from the concerted action of the cationophores and oligomycin is connected with the simultaneous transport of K(+) and its counter ion and that oligomycin plays its role in the uncoupling by facilitating the permeation of Cl(-) through the cristae membrane of the mitochondria.
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