248
`
`SUMMARY
`
`1962
`F. S. STEVEN .AND G. R. TRISTRAM
`Profeeeor Otteaen and hia colleagues for their hoepitality at
`the Carlaberg Institute.
`Our thanks are also due to the British Gelatine and Glue
`Reeearch Aseociation for financial support, and to Dr A.
`Courta for the gift of dinitrophenyl-amino acids.
`
`1. Compounds containing N-terminal residues
`have been shown to be present in preparations of
`soluble collagen which account for most if not all
`the reaction of fl.uorodinitrobenzene with «-amino
`groups.
`2. The significance of the residues has been dis(cid:173)
`cueeed in relation to the known molecular weight
`of collagen as determined by physical means. The
`terminal residues are thought to be due to non(cid:173)
`protein nitrogen firmly attached to collagen and
`not to belong to collagen as true N-terminal
`residues in the usually accepted meaning of the
`term • N -terminal residue•.
`3. The thermal conversion of collagen into
`gelatin has been shown to take place as a physical
`diBBOCiation with tha minimal rupture of peptide
`bonds.
`We wish to thank Imperial Chemical Induatriee Ltd. for
`a ~rch Fellowship (to F.S.S.) and the Department of
`Scientific and Industrial ~rch for a Senior O.E.E.C.
`Vi&iting Fellowship which enabled one of ua (F.S.S.) to
`vi.lit the Carlaberg Laboratory. We are very grateful to
`
`REFERENCES
`
`Bowes, J. H., Elliott, R. G. & Moae, J. A. (1957). In
`Ocmmctive Ti&atu Sympoai.um, p. 264. Ed. by Tun(cid:173)
`bridge, R. E. d al. Oxford: Blackwell Preea.
`Courta, A. (1954). Bioc/&em. J. 58, 70, 74.
`Courts, A. (1959). Bioc/&em. J. 73, 596.
`Fraenkel-Conrat, R. & Singer, B. (1954). J. AmO'. cl&em.
`Boe. 76, 180.
`Reyna, K. & Legler, G. (1957). Hoppe-Beyl. Z. 308, 165.
`Reyna, K. & Legler, G. (1958). In R ~eenl Advamu in
`Gdalim and Give RuearcA, p. 186. Ed. by Stainaby, G.
`London: Pergamon Pre..
`Orekhovitch, V. N. & Shpikiter, V. 0. (1957). In Ocm-(cid:173)
`maive T~ Bynvp<mvm, p. 281. Ed. by Tunbridge,
`R. E. t.l al. Oxford: Blackwell Preee.
`Phillips, D. M. P. (1956). Biocl&em. J. 60, 403.
`Sanger, F. (1945). Biocl&em. J. 39,507.
`Steven, F. S. & Tristram, G. R. (1962). Bioc/&em. J. 83,240.
`Ward. A.G. (1960). J. Boe. Lt-alh. Tr. Chem. 44,505.
`
`Biocheffl. J. (1962) 83, 248
`
`The Degradative Metabolism of L-Cysteine and
`L-Cystine in vitro by Liver in Cystinosis
`
`BY A. D. PATRICK
`Inatit . . of Ohild HwUh, H08pital,Jor Sid: <Jhildrm, Gnat Onnond Strut, London, W.O. l
`
`(Received 13 Norember 1961)
`
`Since Abderhalden (1903) first detected wide(cid:173)
`spread storage of crystalline cyetine in the tissues
`of an infant at autopsy, and noted the familial
`incidence of the disorder, an increasing number of
`C8888 have been reported in which cyetinoeis has
`been diagnosed by poet- and ante-mortem exami(cid:173)
`nation (Lignac, 1924; McCune, Mason & Clarke,
`1943; Worthen & Good, 1958). The 8880Ciated
`clinical features of this disease include vitamin D(cid:173)
`reei.stant rickets, glucoeuria, generalized hyper(cid:173)
`amino aciduria, and acidosis, symptoms which
`characterize a group of dieeasee based on the
`failure of reabeorptive and base-conserving func(cid:173)
`tion of the renal tubules, and often referred to as the
`Fanconi syndrome
`(Fanconi, 1936). Several
`hypotheses seeking to explain cyetine storage and
`to relate this to the Fanconi syndrome have been
`put forward .. Bickel et al. ( 1953) reported increased
`concentrations of a number of plasma amino acids
`in cyetinoeis, and euggeeted that theee resulted
`
`from a generalized failure of their normal in(cid:173)
`corporation into proteins. The intracellular deposi(cid:173)
`tion of the poorly soluble cyetine and the renal
`anomalies were considered to be manifestations of
`this primary defect. However, 8888ntially normal
`plasma amino acid concentrations have been
`found by several authors using chromatographic
`methods (Dent, 1947; Evered, 1956; Brigham,
`Stein & Moore, 1960), and theee are now generally
`accepted as proof that the amino aciduria is of
`renalorigin.
`·
`A second suggestion propo888 a specific enzymic
`defect in cyeteine-cystine metabolism, leading to
`storage of the resultant exceee of cyetine in the
`tissues and consequent nephrotoxic effects. The
`reduction of cystine to cysteine by cyetine re(cid:173)
`ductase has been considered the most probable site
`of such a defect (see Worthen & Good, 1958). Since
`no investigation of theee latter proposals had been
`reported, a comparison of cyetinotic- and normal-
`
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`CYSTEINE-CYSTINE METABOLISM IN CYSTINOSIS
`Vol. 83
`Slioee were cut free.hand from frozen blocks, and aua(cid:173)
`liver preparations, with respect to the degradation
`pended in the cold buffer to be used in the particular 11888y.
`of cysteine and cystine, was undertaken. Particu(cid:173)
`Suitable slices, selected for translucency, were collected
`lar attention has been given to the enzymic
`into bundles and blotted before transfer to incubation
`reduction of cystine.
`flaak.a. In slioe experiments, ti8811e-dry weights from indi(cid:173)
`Recently, deficiencies in the reduction of cystine
`vidual flasks were determined at the end of the incubation.
`by two different enzyme systems of blood have
`period by removal of the total contents (washing ~th
`been reported in cases of cyatinosis. Worthen &
`water if neceesary to remove small fragments), to which
`Good (1961) concluded that cystine reductaee
`was then added an equal volume of 10% (w/v) trichloro(cid:173)
`(NADH1-L-cyatine oxidoreductaee, EC 1.6.4.1)
`acetic acid. After centrifuging, and washing with water and
`with acetone, the reaidues were dried to constant weight at
`was deficient in two cases examined, whereas
`100•. Homogenate (0·5 ml.) waa precipitated with tri(cid:173)
`Seegmiller & Howell (1961) reported cases of low
`ohloroaoetio acid, and washed and dried similarly. The
`activity of a system catalysing the transfer of
`protein content of other enzyme solutions was measured
`hydrogen from reduced glutathione to cystine.
`apectrophotometrically (Warburg & Christian, 1941).
`Neither of these discordant results is supported by
`the present study.
`
`MATERIALS
`
`.A~ apuimena. 'l'he following Ca&M of cyaiinoai& were
`studied: Caae 1, female, aged 11 yr.; Caae 2, male, aged
`8 yr.; Caae 3, female, aged 7 yr. Ti8811es for comparison
`were obtained from two caees of congenital heart diaeaae in
`which no primary metabolic abnormality had been
`apparent. Theee are referred to aa Normal I (female, aged
`8 yr.) and Normal 2 (male, aged 6 yr.). Autopsies were
`performed within 2 hr. after death. Portione of liver and
`kidney were pl&ced in polye~hyleue bags aml frozen im•
`mediately in crushed t101id CO1 • Thereafter, specimens were
`stored at -25°. Only small eamples of cystinotio kidney
`were obtained, their uae being limited to teeta for cystine
`reduction.
`CMmicau. The following chemicals were obtained from
`the sources indicated: NA.DR. (disodium salt), NADP
`(monoeodium salt), pyridonl phoephate and oc-oxoglutario
`acid, from the Sigma Chemical Co.; glu~ 6-phoephate
`(disodium salt), NADPH1 (monoeodium salt) and L·
`glutamio acid, from the British Drug HoU888 Ltd.; L(cid:173)
`oyatine, L-cyateine, L-cysteio acid, reduood glutathione
`(GSH) and NAD from L. Light and Co. Ltd.; L-cyateine(cid:173)
`sulphinio acid, from the California Corp. for Biochemical
`Reeearoh, Loe Angeles. All other reagenta were of AnalaR
`grade where obtainable.
`
`METHODS
`
`Preparaticn of acetone-dried lir,er powder
`Chopped frozen liver (10 g.) was homogenized by grind(cid:173)
`ing with aand and water (20 ml.) at 0°. After standing in
`the cold for 1 hr. with OOC&8ional stirring, the BUBpenaion
`was filtered through surgical gauze. and acetone (2 vol.) at
`- 5° was added to the filtrate with continuoua stirring.
`The precipitate was oollected by rapid filtration in the oold,
`and washed with cold acetone and finally with ether. The
`powders were stored •~ tlaCUO over magnesium perchlorate
`and liquid paraffin.
`
`Tissue homogtl'lalu and alices
`Homogenates (uaoally 20%) were prepared in the
`appropriate ioe-oold media with a Potter-type glaa homo(cid:173)
`genizer for 1 min. at about 700 rev./min. The layer of fat
`obtained from oertaio specimens after oentrifuging at
`800g for 15 min. at 2° could be removed almost completely
`by screening through aeveral layers of surgical gauze.
`
`Enzyme preparations
`Glucoee 6-phoaphate dehydrogenaae (D-glucoee 6-phoe(cid:173)
`phato- NADP oxidoreduot46e, EC l.l.l.49) woa prepared
`from yeast by the method of Kornberg (1950), and &888yed
`according to Kornberg & Horecker (1955). The final solu(cid:173)
`tion hAd t.n M!tMty of 30 unit.a/ml. Yeast glutathione
`reductaae [NAD(P)R.-glutathione oxidoreductaae, EC
`1.6.4.2) was prepared and &888yed according to Racker
`(1955). The preparation used was that obtained at~~ first
`ethanol-precipitation stage, and po118811118d an act1v1ty of
`approximately 20 000 units/ml. Oxidu.ed glutathione
`(GSSG) used in the reductaae &88&Y waa prepared by the
`method of Rall & Lehninger (1952).
`
`Enzyme a81Jaya
`Cy41tm rorJucl<we. The Botivity of oyatine reduotlleo in
`acetone-dried liver powders was estimated by the initial
`decrea.se in NADR. extinction at 34-0 m,- in the presence of
`cyatlne Added u a aaturated aolutlon in either 0-02M(cid:173)
`phoephate buffer, pH 6·5, or 0-lM-tria buffer, pH 8-2.
`Under these conditione t he solubility of cyat,ine at 22° was
`11·6 and 43·6 mg./ 100 ml. in the phosphate and trie
`buffers respectively, as determined by the method of
`Sullivan, Besa & Howard (1942). An extract of acetone.
`dried powder (0·3 g.) was prepared in 3 ml. of the appro(cid:173)
`priate buffer, and dialysed overnight in the cold against the
`same buffer. Clear extracts were obtained by centrifuging
`for 30 min. at 6000g. Kidney extTacta were &88&yed
`Bimilarly. Cyatinotic kidney, owing to its tibroua nature,
`could not be homogenized satisfactorily in the Potter
`homogenizer. Extracts were prepared by grinding l g.
`with cold 0-0211-phoephate buffer (pH 6·5, 3 ml.) and sand.
`After dialyaia for 40 hr. against this buffer, clear extracts
`were obtained by centrifuging at 3000g for 20 min.
`Alternative methoda of estimation were used for liver
`homogenates, which were dialysed in cellophan tubing
`(5 mm. diam.) for 40 hr. in the cold against the buffer to be
`In the first method, homogenate in
`used in the &888Y-
`phoephate buffer, pH 6·5, was shaken anaerobically in
`Warburg fiaaka for 2 hr. with NADR.andt101id cyatine, and
`the in~ of thiol groups in eamples deproteinized with
`10% trichloroaoetio acid (0-5 vol.) waa determined by
`titration with 5 mN-iodine. The starch-iodine end point
`was detectt,d on an EEL Micro-titrator (Evane Electro(cid:173)
`eelenium Ltd.). In the second method, homogenate was
`incubated in tria buffer, pH 8-5, containing NADR. and
`oyatine, and NADR. extinctions at 340 mµ. were measured
`
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`
`1962
`
`A. D. PATRICK
`at zero time and after 10 min. with samples which had
`was replaced by magnesium chloride (Pirie, 1934). The
`Warburg flasks were flushed continuously with 0 2+001
`been deproteinized by the addition of 95% ethanol
`(l vol.) followed by 10% (w/v) sodium sulphate (0·l vol.).
`(19: 1). At zero time 0·4M-cysteine (0·2 ml.), pH 7·4, was
`A 5 mM-cystine soln. used in theee tests was prepared by
`added from the side arms, and the fl.asks were shaken at
`di880lving L•cystine (30 mg. of free baee) in N-sodium
`37°. When cyetine was need as substrate this was added as
`hydroxide (1 ml.) and adding 0·05M-tris, pH 8·5 (5 ml.);
`the solid (5 mg.) directly to the main compartments at the
`the solution was readjusted to pH 8·5 with N-hydrochlorie
`beginning of the experiment. After 2 hr., inorganic sul(cid:173)
`acid, and diluted to 25 ml. with water. Solutions prepared
`phate in the deproteinized medium (i.e. after addition of
`in this way were stable for at least 4 hr.
`5 ml. of 10% trichloroacetic acid) was determined by a
`modification of the method of Cuthbertson and Tompeett
`Syatem reducing eystim in the pruenu of reduud gluJa.
`(Maw, 1954). In later experiments the method of Dodgson
`thiom. Liver homogenate (20%) in 1·15% (w/ v) potas(cid:173)
`sium chloride was dialysed for 24 hr. in the cold against
`(1961) was found to give more reproducible results.
`0· ht•tris buffer, pH 7·6. The incubation medium, added to
`Oridalicn of L-cysteimsulphinau. The metabolism of L(cid:173)
`W&rburg fl.asks, contained in a final volume of 5 ml.:
`cysteinesulphinate by liver slices in the presence of a:•
`0·lM-tris, pH 7·6 (l ml.); 0·5M-glucose 6-phoephate
`oxoglutarate was measured in the following medium:
`O· l M-phoephate, pH 7·4 (2 ml.); slices (approx. 100 mg.
`(0·2 ml.); 0· l M-magnesium chloride (0·2 ml.); glucoee
`6-phoephate dehydrogenaee
`(10 units); 6 mM-NADP
`fresh wt.); 0·376M-L-cysteinesulphinate, pH 7·4 (0·2 ml.);
`(0·1 ml.); l 0mM-GSH (0·2ml.); mM-pyridoxal phosphate
`water (2 ml.); added to Warburg fl.asks. The gas phase was
`air, and centre wells contained 10% (w/ v) potaaeium
`(0·l ml.); homogenate (0·5 ml.); solid eystine (5 mg.).
`hydroxide (0·2 ml.). After equilibration for 10 min. at
`Side arms contained 0·2 ml. of a zinc acetate reagent (see
`below) to trap any hydrogen sulphide liberated. At zero
`37°, 0-25M-a:-oxoglutarate, pH 7·4 (0·2 ml.), was added
`time, a sample (2 ml.) of medium was deproteinized with
`from the side arm, and oxygen uptake followed for 2 hr.
`10% trichloroacetic acid (1 ml.), and the clear supernatant
`The fl.ask contents were treated with cold 10% trichloro(cid:173)
`acetic acid (5 ml.), and, after centrifuging, pyruvate waa
`(2 ml.) obtained after centrifuging was titrated with 5 mN•
`iodine. Incubation fl.asks were flushed with nitrogen, and,
`estimated by the method ofFriedemann & Haugen (1943).
`after shaking for 2 hr. at 37°, the iodine titration for thiol
`Inorganic sulphate was estimated according to Dodgson
`groups was repeated. Trapped hydrogen sulphide was
`(1961). Glutamate was estimated chromatographically.
`estimated as in the assay of oyeteine desulphydrase.
`Superno.tont (2 ml.) was evaporated to dryn688, and the
`Cyauim desulphydrase. The assay medium, consisting
`residue was extracted with water (0-5 ml., or O· l ml. in
`of 0·067M-phoephate buffer, pH 7·4 (1 ml.), liver homo(cid:173)
`experiments in which either a:-oxoglutarate or cysteine(cid:173)
`sulphlnate wa.s omitted from the medium). Samples
`genate, 20% in 0·9% sodium chloride, (1 ml.) and mM•
`pyridoxal phosphate (0· l ml.), was added to the main com(cid:173)
`(30µ1.) were chromatographed in butan-1-ol-acetio acid(cid:173)
`partments of Warburg fl.asks. Side arms contained 0·l M(cid:173)
`water (12:3:5, by vol.) on Whatman no. 2 paper. Marker
`cysteine, pH 7·4 (0·2 ml.), and centre wells a reagent
`solutions containing 10, 20 and 30µg. of glutamate were
`run simultaneously. After spraying with ninhydrin, the
`(0·2 ml.) consisting of hydrated zinc acetate (6·0 g.),
`hydrated sodium acetate (1·7 g.) and sodium chloride
`approximate concentrations of glutamate in test solutions
`(0·005 g.) di880lved in water (100 ml.). Flasks were flushed
`were estimated by visual comparison. This was found to
`with nitrogen, and shaken for 90 min. at 37°. p-Amino(cid:173)
`give estimates in fair agreement with those obtained by
`NN-dimethylaniline [0·3 ml. of a 0·05% soln. in 20%
`spot elution ~nd oolour measurement.
`(w/v) sulphuric acid} followed by 1 drop of 10% (w/ v)
`Gl'lllaJhiom reductase. This was assayed essentially
`ferric chloride in 0·lN-hydrochloric acid was added to the
`according to the method of Racker (1955). Acetone-dried
`centre well, and t he resulting methylene blue solution,
`liver powder (50 mg.) was extracted with ooltl water
`after colour development for 1 hr., was washed out and
`(l ml.) for 1 hr. After centrifuging, the solution was
`diluted to 25 ml. with water. Extinctions at 630 mµ were
`dialysed overnight in the cold against 0·2M-phosphate,
`measured in 1 cm. cuvettes. An aq. hydrogen sulphide soln.,
`pH 7·6, and recentrifuged.
`standardized with sodium arsenite, waa used to prepare a
`All spectrophotomet ric measurements were made in
`standard curve for the range 10-100 µg. of hydrogen sulphide.
`1 cm. silica cells with a Unicam SP. 500 spectrophoto(cid:173)
`Desulphurizaticn of cystim. This system refers to the
`meter (Unicam Instruments Ltd.).
`NADRt-dependent reduction of cystine to eysteine,
`followed by the liberation of hydrogen sulphide by cysteine
`desulphydrase [L-cysteine hydrogen sulphide-lyase (de(cid:173)
`aminating), EC 4.4.l.l}. The hydrogen sulphide, estimated
`as described above, gave a measure of the overall reaction.
`Alcohol dehydrogenase (alcohol-NAD oxido-reductaee,
`ECI.1.1.1), contained in the liver preparations, was used
`liver homogenate was
`to regenerate N ADH1 • A 10%
`prepared in 0·9% sodium chloride and dialysed overnight
`in the cold against 0·067M-phoephate buffer, pH 7·4.
`Decarboxylalicn of L-eysteale and L-eysteimsulphinau..
`This was determined for liver extracts according to the
`method of Hope (1955).
`Oxidalicn of L-eysuim and L-cy8tim to inorgan~ sulphau.
`Liver slices were suspended in 5 ml. of bicarbonate-saline
`(Krebs & Henseleit, 1932), in which magnesium sulphate
`
`The reduction of cystine by cystine reductase.
`A comparison of the activities of cystine reductase
`in liver homogenates and kidney extracts, and in
`acetone-dried liver powders of the cystinotic and
`normal cases, is shown in Table I. Tests were made
`after the tissue samples had been stored for
`2 months a t - 25°, except for cystinosis Case I
`(Expt. D), which had been stored for 3 months.
`The activities of the cystinotic- and normal-tissue
`preparations were in good general agreement in
`ea.ch experimental series, with different conditions
`
`RESULTS
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`
`CYSTEINE--CYSTINE METABOLISM IN CYSTINOSIS
`
`251
`
`Table I. Oyaeine-redudalle activity of liver and kidney preparaewna
`Silica cuvettes of l om. light path cont&ined 2·5 ml. of & a&tur&ted soln. of oystine in 0·02M-phoeph&te buffer,
`pH 6·5 (Expt. A), or in 0-1 M-tris buffer, pH 8·2 (Expt. B); acetone-dried liver-powder extract (0-5 ml.) <(cid:173)
`Methods eection); and 1·4 DlK-NADHs (0·1 ml). Extinctions at 340 mµ were me&aured at l min. intervals for
`10 min. at room temperature (22°). Blank 888&y& contained buffer instead of buffer-oyatine soln. Kidney extract
`(0·5 ml.) (see Methods eection) was aaa&yed similarly in 0·021l-phoaphate, pH 6·5 (Expt. C). In Expt. D, incuba(cid:173)
`tion media contained liver homogenate (l ml.); 0-05M-tris buffer, pH 8·5 (l ml) ; 5 mM-cyatine, pH 8·5 (2 ml.),
`or water; 3 mM-NADH1 (0-2 ml.); in 5 ml. final volume. Incubation temperature waa 30°. Extinctions at
`340 mµ of samples deproteinized at zero time &nd &fuir 10 min. (see Methods section) were meaaured. In Expt. E,
`media contained liver homogenate (2 ml.); 0·02H-phoeph&te, pH 6·6 (2 ml.); 14 mH-NADHs (O·l ml.); water
`(l ml.); solid cyatine (6 mg.); &dded to Warburg fl.asks. Flaab were shaken &n&erobically at 37° for 2 hr., and the
`increase of thiol groups w&a determined by titration with 5 mN-iodine.
`Activity
`( -t:..Ew;/mg. dry wt.fhr.)
`
`Activity
`( -t.E .. 0/mg. of proteinfhr.),
`Acetone-dried.-liver(cid:173)
`powders preparation
`
`Case
`Cystinoeis l
`Cystin08ia 2
`Cystinoais 3
`Norm&! l
`Norm&! 2
`
`Expt.A
`0·052
`0·039
`0·038
`0·047
`0·044
`
`Expt. B
`0-055
`0·031
`
`0·050
`
`and methods of 8888.y. It wM found that the
`cyatinotic homogenates released thiol groups in the
`absence of added cyatine when such preparations
`were dialysed for only 24 hr. This activity,
`which was completely dependent on the addition
`in BQme homogenate preparations
`of NADH2 ,
`amounted to 60 % of that obtained by full stimu(cid:173)
`lation with added cystine. Dialysis for 40 hr. in
`cellophan tubing (5 mm. diam.) reduced this
`endogenous activity almost to zero without causing
`undue lOBB (not more than 10 %) of overall cystine(cid:173)
`stimulated activity. It appears certain from these
`experiments that the high endogenous activity
`was due to stored cyatine, low residua.I concentra(cid:173)
`tions of which would be sufficient to saturate the
`enzyme in the most active of the preparations
`tested. Fig. I shows the effect of varying concen(cid:173)
`trations of such a preparation in assay media
`containing the lowest concentrations of cystine,
`i.e. when cystine was added as a saturated solution,
`pH 6·5. The specific activities given in Table 1
`were confined to determinations carried out in the
`linear range of the activity curves. Fig. I also
`illustrates the specificity of cystine reductase for
`NADH1 , an equivalent a.mount of NADPH,
`failing to a.ct as replacement, and shows the com(cid:173)
`plete loss of activity of a boiled preparation.
`is
`Evidence that cystine-reductase activity
`norme.1 in cystinotic liver
`(stored frozen for
`14 weeks) was a.lso obtained from determinations
`of the desulphuriza.tion of cystine by homogen(cid:173)
`ates. The liberation of hydrogen sulphide from
`cystine was completely dependent on additions of
`NAD and, with one exception (cyatinosis Caae 3, in
`
`Thiol groups
`formed (µmolea/g.
`dry wt./hr.).
`Liver-homogenate
`preparation
`Expt. E
`25·5
`33·0
`29·0
`31·5
`25·5
`
`Kidney(cid:173)
`extract
`preparation
`Expt. 0
`0·019
`0·021
`
`0·024
`
`Liver(cid:173)
`homogenate
`preparation
`Expt. D
`0-024
`0-029
`0·024
`0·026
`0·021
`
`0-3
`
`0-1
`
`0
`
`2
`
`4
`
`8
`6
`Protein (mg.)
`
`10
`
`12
`
`14
`
`Fig. l. Cyatine-reduct&ee activity of acetone-dried liver
`powder (cyatinosia Case 3). Varying &mounts of enzyme
`were aaaayed under the experimental conditions of Expt. A,
`Table l. O, Complete system; • • oyatine omitted; 6,
`enzyme soln. boiled for 2 min.; D, NADHs replaced by
`NADPlis; • • NADHs omitted.
`
`which a low endogenous activity was observed), a
`NAD-linked oxidizable substrate. In the experi(cid:173)
`ments listed in Table 2, ethanol was used as
`oxidizable substrate for
`the regeneration of
`NADH, by alcohol dehydrogenase of the liver
`homogenates. The results a.re
`interpreted as
`showing a norme.1 reduction of cystine to cyateine
`by cystine reductase of cyatinotic liver, followed by
`the liberation of hydrogen sulphide froµi cysteine
`by cysteine desulphydrase. The slight activity of
`
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`
`

`

`Table 2. Deatdphurization of L-cyatine by liver
`homogenai,u
`
`1962
`
`Media contained 0-067M-ph08J>hate, pH 7·4 (1·5 ml.);
`liver homogenate (0·5 ml.) (see Methods section); 3 mK•
`NAD
`(0·l ml) ; 2 lllll-pyridoxal phosphate (0·l ml.);
`ethanol (0·l ml); solid cystine (5 mg.); added to Warburg
`fiaeka. The trapping and estimation of H.,s were carried out
`as for the aeeay of cysteine deeulphydraae (see Methods
`section). Anaerobic incubation was at 37° for 4 hr., with
`constant shaking.
`
`Activity (µmoles of 11-8/g. dry wt.
`of liver homogenate/hr.)
`
`Complete
`system
`7·9
`11·8
`7·4
`8·2
`
`Ethanol
`omitted
`0
`2·2
`0
`0
`
`Cystine
`omitted
`O·l
`0·3
`0
`0
`
`Case
`Cyetinoeie 2
`Cystinoeis 3
`Normal 1
`Normal 2
`
`252
`A. D. PATRICK
`the cystinotic samples observed in the tests from
`which cystine was omitted was attributed to
`residual stored tissue cystine.
`The reduction of cyatine by reduced gltdathione.
`In the presence of reduced gluta.thione, cystine is
`converted into cysteine. Liver homogenates stimu(cid:173)
`late this reaction, and further metabolize part of
`the cysteine formed, mainly through the desulph(cid:173)
`ydrase reaction under anaerobic conditions. With
`low concentrations of gluta.thione the overall
`system requires glutathione reductase, coupled
`with a reaction which regenerates NADPffs. In
`the experiments on liver homogenates, this latter
`requirement was met by the addition of the glucose
`6-phoephate-dehydrogenase system. Homogenates
`poaeeaaed sufficient glutathione-reductase activity
`to maintain maximum concentrations of reduced
`glutathione throughout the incubation period.
`A comparison of the activities of glutathione re(cid:173)
`ductase in cystinotic and normal liver shows good
`agreement of values independent of the period of
`storage of the frozen tissues (Table 3).
`Table 4 shows the requirements of the cystine(cid:173)
`reduced glutatbione system for cystine, reduced
`glutathione, a NADP-linked oxidizable substrate
`(glucoee 6-phosphate) and glutathione reductase.
`To account for the extent of spontaneous non(cid:173)
`enzymio reduction of oystine by reduced glut•
`athione in the overall reaction, it was necessary to
`employ a composite system in which a glutathione(cid:173)
`reductase preparation was substituted for liver
`homogenate in these experiments. A comparable
`concentration of roduood gluto.thiono woe thereby
`
`Table 3. Gltdathione-reductaae activity of liver
`Microouvettee of 1 cm. light path contained: M-phos(cid:173)
`pbate buffer, pH 7·6 (0-05 ml.); mx-NADPH. (0·05 ml.);
`1 % (w/v) serum albumin (0·05 ml); extract of acotono(cid:173)
`dried liver powder (0·05 ml.) (see Methods section); water
`(0-2 ml.); 30 mx-GSSG (0·05 ml.) or water. Extinctions at
`34-0 m,i were meiMJiited At 30 eoo. i.utervale for 5 m.ln. At
`room temperature (22°).
`Activity
`( - br.E .. 0/mg, of
`protein/min.)
`0·106
`0-121
`0-110
`0·105
`0-088
`
`Period of
`tissue storage
`14 months
`9 months
`3 weeks
`11 months
`6 months
`
`Cyetinoeie 1
`Cyatinoeis 2
`Cyetinoeie 3
`Normal 1
`Normal2
`
`Table 4. Reduction of cyatine in the presence of reduced gltdathione
`
`A. F or experimental details of the overall reaction (enzymio + spontaneous), see Methods section. B. Spon(cid:173)
`taneous reaction; the medium contained: 0·l:li1-trie buffer, pR 7·6 (l ml.); 0-5:M-glucoee 6-phosphate (0-2 ml.);
`0-lM-MgCI. (0·2 ml); gluooee 6-phoepbate debydrogenaee (0-2 ml.= 10 unite); 5 Illll•NADP (0-1 ml); 10 mil•
`GSH (0·2 ml.); glutathione reductaee (0-05 ml. = 1000 units); solid cyetine (5 mg.); in a final volume of 5 ml.,
`contained in Warburg flasks. The experiment was otherwise performed as for (A). Results are expre.,d as
`changes occurring in 2 hr. at 37°.
`Complete system
`
`Difference in 5 mll"•I, required
`(ml./ml. of reaction medium)
`
`Glucose
`6-phoe-
`Period
`of storage Complete Cystine
`phate
`GSH
`of liver
`system
`omitted omitted omitted
`6 months + 1·01
`0
`+0-02
`+ 0·03
`+ 0·82
`+0·03
`-0·08
`5 weeks
`+ 0·03
`11 months + 0·31
`-0·03
`+ O-Oi
`+0-01
`3 months + 0·86
`+0·03
`+0·02
`+ 0·02
`
`Glut.a-
`thione
`reductaee
`omitted
`
`Thiol groups formed
`
`HaS*
`liberated
`(µmoles/
`ml.)
`0·18
`0-22
`0·27
`0-31
`
`Total
`(µmoles/
`ml.)
`5·20
`4·37
`1·85
`4·60
`
`Correctedt
`(µmolee/mf"
`dry wt. 0
`liver homo-
`genate/ml.)
`0·66
`0·63
`0·03
`0-61
`
`+ 0-34
`
`+0-01
`
`+0-02
`
`0
`
`0
`
`0
`
`M0t
`
`• No HaS was liberated from incomplete media.
`t Calculated after subtraction of mean value for thiol groupe formed in the spontaneous reaction.
`t Range 1-43---1-95 (mean 1-71, from four estimations).
`
`A
`Case
`Cystinoeie 2
`Cyetinoeie 3
`Normal 1
`Normal2
`B
`Spontaneous
`reaction
`
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`
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`

`Vol. 83
`
`253
`production such as is seen with the stimulation
`effected by the addition of cysteine or cystine.
`The high concentrations of endogenous sulphate
`produced by cystinotic specimens, and the apparent
`failure of cystine to stimulate this production, may
`be explained by a maximally stimulated formation
`of sulphate already occurring in the presence of
`stored cystine. The ready availability of intra(cid:173)
`cellular cystine in these specimens could aleo
`account for the endogenous production of sulphate
`in greater amount than that derived from cystine
`additions to normal liver.
`Further evidence of the normal oxidation of
`cystine in cystinosis was derived from the deter(cid:173)
`mination of urinary inorganic sulphate in two cases.
`The values obtained were 13·0 and 11·3 mg.
`of inorganic sulphate Sfkg. body wt./24 hr., com(cid:173)
`pared with 9·4 and 14·6 mg. of sulphate Sfkg./24 hr.
`for two normal children of a.bout the same body
`weight.
`2'he meiaboli.am of L-cyateineaulphinau. A balance
`study of cysteinesulphina.te metabolism in liver
`slicea is summarized in Table 6. Liver specimens
`
`Table 5. Oridation of L-cyateine and L-cystiM to
`inorganic atdphau by litJer slices
`Experiment&! detail.a are given in the Method.a eection.
`Inorganio IU!phate production after 2 hr. at 37° was
`m~smroo.
`
`Activity (pg. of IU!phate S/100 mg.
`dry wt. of liver Blioee)
`
`CYSTEINE--CYSTINE METABOLISM IN CYSTINOSIS
`maintained in aaaay media that were otherwise
`identical, eo that the difference in cystine reduction
`in the two systems gave a measure of the stimu(cid:173)
`lating effect of liver homogenate. Result.a for the
`spontaneous reaction (which coincided approxi(cid:173)
`mately with the m ean values for four separate
`determinations) are given in Table 4B. Sub(cid:173)
`traction of this value for thiol groups formed from
`those obtained for the overall reaction in homo(cid:173)
`genates (Table 4A) gave good agreement for the
`enzymic component of cystinotic- and normal-liver
`specimens which had been stored frozen for up to
`6 months. With further storage considerable loss of
`activity may occur, a 95 % loss being observed for
`the normal sample stored for 11 months.
`In view of the normal capacity of cystinotic liver
`for glutathione--cysteine transhydrogenation and
`glutathione reduction in t>itro, it was of some interest
`to determine the concentrations of reduced glut(cid:173)
`a thione in liver specimens, since a failure to main(cid:173)
`tain this (caused indirectly, e.g. by a deficiency of
`NADPRt) could impair the subsequent reduction
`of cystine. EetiJiltltiona were made on sulphosali(cid:173)
`cylic acid extracts by the yeast-glyoxalsse method
`of Woodward (1935). The mean values obtained
`were l ·5 mg. and l ·3 mg. of reduced glutsthione/g.
`wet wt. of liver from the cystinosis and normal
`<.'-Mefl respectively .
`The oa:idatwn of cyateine and cystine to inorganic
`aul,phate. Slices of cystinotic liver were found to
`possess a normal capacity for the complete oxid(cid:173)
`ation of cysteine, and probably of cystine, to in(cid:173)
`organic sulphate (Table 5). In all cases the liver
`specimens had been stored frozen for 2 months
`before testing. The amount of endogenous sulphate
`derived from normal specimens was in the range
`reported by Maw (1954) and Med.es (1939) for rat(cid:173)
`liver slices, and is ascribed to diffusion of existing
`intracellular sulphate rather than to a metabolic
`
`Subetrate
`Case
`Cyetinoeia l
`Cyetinoeis 2
`Cyetinoeis 3
`Normal l
`Normal 2
`
`Cyeteine
`
`Cyatine
`
`l!M)
`151
`217
`129
`107
`
`72
`52
`81
`43
`3lS
`
`None
`
`60
`54
`76
`21
`16
`
`Table 6. Melaboli.am of L-cyateineaulphinate in liver slice.a
`Experimental detail.a are given in the Method.a eection. Values refer to changes occurring in 2 hr. at 37°.
`
`Caee
`Cystinoaia 2
`
`Normal l
`
`o.uptake in
`exoees of
`endogenoUB level
`(µg.-atoms of
`0 1/g. dry wt. of
`Glutamat.e
`Sulphate
`Pyruvate•
`liver slices)
`Subetanoe omitted
`61·0
`120·0
`74·5
`117·0
`None
`39·5
`cx-Oxoglutarate
`<3·0
`14--7
`1-7
`13·0
`39·1
`0
`Cyeteineeulphinate
`<3·0
`54·0
`129·5
`76·5
`102·5
`None
`44·8
`cx-Oxoglutarate
`8·8
`25·5
`<3·0
`9.5
`<3·0
`9·3
`33·5
`Cyateineaulphinate
`• Valuee for pyruvate were not expected to be accurate in the presence of cx-oxoglutarate in the conoe1;1trations 11.8ed.
`Values recorded for experiments in which cyi,teineeulphinate wae omitted were moat probably due ent~ly to cx-oxo(cid:173)
`glutarate rather the.n to pyruvate. However, the experiments aerve to show cloee agreement in the net formation of
`pyruvate in the complete systems.
`
`Subetanoee formed
`(p.molee/g. dry wt. of liver elicee)
`
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`
`

`

`254
`
`1962
`
`A. D. PATRICK
`(Worthen & Good, 1958), it was of interest to
`had been stored for 18 weeks. Oxidation of
`cysteinesulphinate to sulphate was stimulated by
`determine the activities ofL-cystes.te decs.rboxylase
`0(-oxoglutarate, and the appearance of glutamate
`and L-cysteinesulphinate decs.rboxyls.se (L•cysteine(cid:173)
`required the presence of cysteinesulphinate, in
`sulphinate carboxy-lyase, EC 4.1.1.29) in the liver
`keeping with a reaction mechanism requiring an
`specimens. Results of these experiments (Table 7)
`initial transamination with the formation of {J(cid:173)
`indicate the normal ability of cystinotic liver to
`sulphinylpyruvate. Stoicheiometric balances were
`decs.rboxylate these substrates, of which cysteine(cid:173)
`not observed, but the overall similarity of results
`sulphinate shows the higher decs.rboxylation rate.
`obtained with cystinotic- and normal-liver speci(cid:173)
`Anaerobic f"eacti