The Intracellular Ratio of Cysteine and Cystine in Various Tissues

Biochem. J. (1967) 105, 891 891 Printed in Great Britain The Intracellular Ratio of Cysteine and Cystine in Various Tissues By J. C. CRAWHALL* AND S. SEGALt Clinical Endocrinology Branch, National In8titute of Arthriti8 and Metabolic Di8ease8, National Institute8 of Health, Bethe8da, Md., U.S.A. (Received 20 February 1967) 1. The cysteine-cystine ratio was measured in rat kidney cortex, diaphragm, jejunum, liver and brain. 2. This ratio was determined by incubating these tissues in buffer containing [35S]cystine and then homogenizing the tissue in a buffered solution of N-ethyhnaleimide. The products of this reaction were separated by high-voltage electrophoresis and the radioactivity in the cystine and 2-(L-2'-amino- 2'-carboxyethylthio)-N-ethylsuccinimide regions was determined. 3. In these tissues cyst(e)ine was mainly present in the reduced form. 4. After incubation of [35S]cystine with rat jejunal segments it was found that 36% of the cystine in the medium has been reduced. 5. Anaerobiosis, Na+-free media, glucose and high concentrations of cystine and lysine were found not to affect significantly the cysteine-cystine ratio in rat kidney-cortex slices. Numerous studies of the kinetics of amino acid The method of measuring cystine-cysteine ratios transport into various tissues in vitro have been described in this paper has been designed to remove described. One of the requirements of this type of all the cellular enzyme activity as rapidly as study with radioactive isotopically labelled amino possible at the end ofthe tissue incubation period by acids is that the radioactivity measured within the precipitation of the enzymes with trichloroacetic tissue corresponds to the amino acid being studied acid. This prevents any further reduction of intra- and not a metabolite of that amino acid. Investiga- cellular cystine during the isolation procedure. The tions of cystine transport have so far not distin- reoxidation of cysteine was prevented by blocking guished between the intracellular oxidized and the free thiol group with NEM.4 It was found that reduced forms ofthe amino acid. This investigation this reagent reacted much faster with thiol groups was designed to use the active transport mechanism than did iodoacetate. Before true intracellular of various tissues to introduce radioactive iso- cystine-cysteine ratios can be calculated from topically labelled cystine into the tissue and then experiments in which tissues have been incubated the extent of reduction of the labelled cystine was in a buffer containing [35S]cystine, it is necessary measured. to make a correction for the amount of amino Previous studies of the ratio of reduced to acid retained in the tissue that is in the extra- oxidized glutathione and of cysteine to cystine in cellular fluid and has not entered the cells. This certain tissues by colorimetric methods have can be done by assuming that the concentration of indicated that glutathione and cysteine are amino acid in the extracellular fluid is the same as principally in the reduced state (Fujita & Numata, that in the incubating medium. This correction 1938a,b). More than one possible mechanism by was carried out according to the procedure of which this reduction can be maintained have been Rosenberg, Blair & Segal (1961). We have measured described. Cystine reduction can occur by coupling the cystine-cysteine ratio in the medium as well as through glutathione and glutathione reductase to the tissue at the end of the incubation period. In NADH (Pihl, Eldjarn & Bremer, 1957). A direct those cases where no reduction of cystine in the cystine reductase has been described (Patrick, medium occurred, it was assumed that all the 1962) but not characterized and the existence of cystine trapped in the ECF would be in the oxidized such a separate enzyme mechanism of this type has form. If some of the cystine in the medium was been questioned (Black, 1963). reduced it was assumed that the same cystine- ratio would be in the ECF as was * Present address: Professorial Medical Unit, St cysteine present Bartholomew's Hospital, London, E.C. 1. present in the medium. t Present address: The Children's Hospital of Phila- $ Abbreviations: NEM, N-ethylmaleimide; Cys-NEM, delphia, Department of Pediatrics, University of Pennsyl- 2 - (L-2'-amino-2'-carboxyethylthio) - N - ethylsuccinimide; vania Medical School, Philadelphia, Pa., U.S.A. ECF, extracellular fluid; ICF, intracellular fluid. 892 J. C. CRAWHALL AND S. SEGAL 1967 MATERIALS AND METHODS washing the tissue was transferred to filter paper for blotting Isotopically labelled compounds. [35S]Cystine was pur- and weighing. Distribution ratios, the ratio counts/min./ml. chased from Schwarz BioResearch Inc. (Orangeburg, N.Y., of ICF to counts/min./ml. of medium, were calculated U.S.A.) and made up as a 2mM solution in dilute NaOH according to Rosenberg et al. (1961) after extracting the containing about lOpc/ml. Solutions were made frequently tissue amino acid pool with warm water. (about every 2 weeks) since analysis of these solutions by Determination of total tissue water and extracellular space. high-voltage electrophoresis revealed oxidation to cysteic Total tissue water was determined from the difference acid to occur with time. Unless stated otherwise 1OO,]. of between tissue weight after blotting the tissue slices and the this solution was added per flask to give a final concentration weight after drying them in a crucible at 1050 for 24hr. 0-1 mM. When cystine was converted into cysteine by This value was expressed as per cent of wet-tissue weight. dithiothreitol, 50,l. was added to give 0- mM-cysteine. The extracellular space of the tissues was determined by [carboxy-14C]Inulin was obtained from New England the method of Rosenberg, Downing & Segal (1962) with Nuclear Corp. (Boston, Mass., U.S.A.). [carboxy-14C]inulin. Chemicals. Dithiothreitol and NEM were obtained from Use of iodoacetate to block cysteine thiol groups and the rate Calbiochem (Los Angeles, Calif., U.S.A.). Cys-NEM was of [35S]cysteine reaction with iodoacetate. The method of prepared according to Smyth, Nagamatsu & Fruton (1960). Brigham, Stein & Moore (1959) was utilized. After incuba- High-voltage paper electrophoresis. This was carried out tion in [35S]cystine rat kidney-cortex slices were dipped in with a Gilson Electrophoretor model D in 6-8% formic acid 0-9% NaCl soln., blotted and homogenized in a Potter- at 450 at 4000v for 2 hr. with 43 in. Whatman 3MM papers Elvehjem homogenizer for 1 min. in 1 ml. of 0-IM-phosphate (Rosenberg, Crawhall & Segal, 1967). buffer, pH 7-6, containing 100mg. of iodoacetic acid, Under these conditions, cystine migrates on the filter 42mg. ofNaHCO3 and 20mg. ofdisodium EDTA. A 0-1 ml. paper 2 in. further than Cys-NEM and these are both sample was removed at increasing times (5, 10, 20 and separated by 6in. from the more acid products of cystine 30min.) and mixed with 0-1 ml. of 10% trichloroacetic acid. metabolism (cysteic acid, cysteinesulphinic acid and Each of these fractions was then submitted to high-voltage taurine). electrophoresis and the radioactivity in the cystine and Ninhydrin reagent used for staining marker amino acids carboxymethylcysteine regions was determined. As a was prepared as follows. Ninhydrin (4g.) was dissolved in zero-time incubation control three kidney slices were added 400ml. of acetone. Cadmium acetate (0-4g.) was dissolved to 1 ml. of iodoacetate soln. containing 0-02mM-[35S]cystine in a mixture of 8ml. of acetic acid and 16ml. of water. The and homogenized immediately. After 30min. lml. of cadmium acetate solution was then added to the ninhydrin trichloroacetic acid was added and samples of this mixture solution. The resulting reagent could be used for about were submitted to electrophoresis. 3 weeks if stored in a dark bottle. Unstained areas corre- The rate ofreaction of [35S]cysteine with iodoacetate was sponding to cystine and Cys-NEM were cut from the measured as follows: 50,ul. of [35S]cystine (2mM) and 50,u1. papers, placed in glass vials with 10ml. of toluene-based of dithiothreitol (10mm) were added to 0-9ml. of potassium phosphor (Liquifluor; New England Nuclear Corp.) and the phosphate buffer (0-1M, pH7-4). After 10min. iodoacetate radioactivity was determined by liquid-scintillation count- solution (1 ml.) was added, after which 0-1 ml. samples were ing (Tri-Carb liquid-scintillation spectrometer; Packard removed at 0, 5, 10, 20 and 30min., and added to 0-1ml. of Instrument Co.) at approx. 50% efficiency. 6-8% formic acid. Samples (2Opl.) ofeach mixture were sub- Preparation of tissues for studies in vitro. Tissues were mitted to high-voltage electrophoresis and the cystine and obtained from 150g. male Sprague-Dawley rats, which had carboxymethylcysteine regions counted for radioactivity. been fed ad libitum on a Purina rat chow diet. Rate of reacion of cysteine with NEM and the effect of The technique for incubations and study of intracellular kidneyslices on [35S]cystine in the presence of NEM. The rate accumulation of isotopically labelled amino acids was of reaction of cysteine with NEM was studied in a similar essentially that described by Rosenberg et al. (1961) for manner to the previous experiment. [35$]Cystine (50zl., kidney-cortex slices and by Marsh, Rosenberg & Segal 2mM), dithiothreitol (50,u1., 10mM) and potassium phos- (1962) for jejunal segments and diaphragm. Kidney and phate buffer (0-1M, pH7-4; 0-9ml.) were mixed together. liver slices were prepared by a Stadie-Riggs microtome After 6min., NEM (lml., 20mM) in phosphate buffer (0-4mm. thick). (1 ml., 0-1 M, pH 7-4) was added and samples (0-1 ml.) were The following tissues were incubated in Krebs-Ringer removed at increasing intervals of time (0, 1, 2-5, 10min.) bicarbonate buffer, pH 7-4 (2 ml.) (Krebs & Henseleit, and added to 6-8% formic acid (0-lml.).

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