Clinical Science and Molecular Medicine (1975) 48, 509-513.
Methionine adenosyltransferase, cystathionine p-synthase
and cystathionine y-Iyase activity of rat liver subcellular
particles, human blood cells and mixed white
cells from rat bone marrow
JENNIFER ALLSOP
AND
R. W. E. WATTS
Division of Inherited Metabolic Diseases, Medical Research Council
Clinical Research Centre, Harrow, Middlesex
'
(Received 6 December 1974)
Summary
1. Methionine adenosyltransferase (ATP:Lmethionine-S-adenosyl transferase, EC 2.5.1.6),
cystathionine p-synthase [L-serine hydro-lyase
(adding homocysteine), EC 4.2.1.22] and cystathionine y-Iyase [t-cystathionine cysteine-lyase
(deaminating), EC 4.4.1.1] activities were found
only in the cytosol fraction of rat liver cells.
None was found in the mitochondrial or endoplasmic reticulum fractions as judged by the distribution of marker enzymes on a density gradient after
centrifugation of the cytoplasmic fraction of a
liver homogenate, or in a preparation of liver cell
nuclei.
2. Polymorphs, lymphocytes (with admixed
monocytes) and mixed bone marrow white cells
contained no methionine adenosyl transferase,
cystathionine p-synthase or cystathionine )I-lyase
activities.
3. The possible bearing of these results on the
problem of abnormal cystine storage in cystinosis is
briefly discussed.
phages, the polymorphs and cultured fibroblasts
contain abnormal amounts of cystine within
membrane-bounded structures, which resemble
lysosomes (Hummeler, Zajac, Genel, Holtzapple &
Segal, 1970). Fibroblasts from cystinotic patients
also incorporate more [35S]cysteine into cystine
than normal subjects' fibroblasts (Schneider,
Bradley & Seegmiller, 1968; Willcox & Patrick,
1974).
The three sequential steps of the transulphuration
metabolic pathway from methionine to cysteine
are catalysed by methionine adenosyltransferase,
cystathionine p-synthase and cystathionine )I-lyase
respectively; and there is normally little intracellular
oxidation of cysteine to cystine (Crawhall & Segal,
1967; Crawhall & Davies, 1969).
The present work was undertaken to locate the
cysteine-synthesizing enzymes within cells and to
determine if blood or bone marrow leucocytes have
detectable transulphuration enzyme activity.
Methods
Male Sprague-Dawley rats weighing 400-500 g were
killed by cervical dislocation. All the subsequent
manipulations were at Q-4°C. The liver was removed
immediately, washed in sucrose solution (250
mmol/l) and homogenized in another portion of this
medium in a Potter-Elvehjem homogenizer with
three up-and-down strokes of a Teflon pestle. The
homogenate was diluted with sucrose (250 mmol/l)
to give a final concentration of 1 g wet weight of
liver/l0 ml.
The homogenate was centrifuged at 600 gay. for
Key words: cystathionine )I-lyase, cystathionine
p-synthase, cystinosis, methionine adenosyltransferase, transulphuration.
Introduction
Cystinosis is an autosomal recessively inherited
disorder in which the reticuloendothelial macroCorrespondence: Dr R. W. E. Watts, Division ofInherited
Metabolic Diseases, Clinical Research Centre, Watford
Road, Harrow, Middlesex HAl 3UJ.
509
510
Jennifer Allsop and R. W. E. Watts
10 min to produce a supernatant (cytoplasmic
fraction), and a pellet which contained largely
nuclei, unbroken cells and plasma membranes.
Continuous gradients were prepared with a
gradient former. To prevent pellet-formation on the
bottom of the tube, a layer (3'5 ml) of 82% (w/v)
sucrose solution (2,40 rnol/l) was placed in the
bottom of a 50 ml polypropylene centrifuge tube, a
40 ml continuous gradient of sucrose concentrations
(1'66-0'25 mol/I) formed above it and the system
left to equilibrate at 4°C overnight. A portion (5 ml)
of the cytoplasmic fraction was carefully layered on
top of the gradient and the tubes were centrifuged
for 2 h at 4°C and 1350 g (r av. 25 em) in a swing-out
rotor. These conditions are a modification (Danpure,
1971) of those described by Rahman & Lindenbaum
(1964). The gradient was fractionated by piercing a
hole in the bottom of the tube and collecting 16 x 3
rnl aliquots. Sucrose concentrations were measured
with the Abbe refractometer.
Granulocytes were separated from human blood
as described by Westwick, Allsop & Watts (1972).
This involved centrifuging dextran-treated plasma
on a layer of a bovine serum albumin solution. The
polyrnorphs sedimented through the bovine serum
albumin, and lymphocytes with admixed monocytes
remained at the interface. The polymorph- and
lymphocyte-containing fractions were collected and
any residual erythrocytes lysed by hypotonic shock.
Marrow mixed white cells were prepared from rat
bone marrow by washing out the cells from the
femora with NaCI (154 mmol/l) and centrifuging the
suspension at 100 gav. for 5 min. The cells were
resuspended in NaCI (154 mrnol/l) and the erythrocytes were removed by hypotonic lysis. The
cells were again resuspended in NaCI (154 mmolfI).
The white cells were separated by centrifugation
(100 gav,) for 5 min. All of the cell preparations were
immediately disrupted by ultrasonic vibrations and
assayed immediately.
Nuclei were prepared by the method of Blobell &
Potter (1966). The final preparation had active RNA
polymerase (1,7 pmol min -1 /lg-1 of DNA) and the
assays of the transulphuration enzymes were performed on aliquots containing 37'4 ug of DNA.
Lactate dehydrogenase (L-Iactate:NAD oxidoreductase, EC 1.1.1.27) was assayed by the method of
Plummer & Wilkinson (1963), and cytochrome c
oxidase (ferrocytochrome c: oxygen oxidoreductase,
EC 1.9.3.1) by a modification of the method of
Cooperstein & Lazarow.(1957) similar to that of
Appelmans, Wattiaux & de Duve (1955). Arylsulphatase (arylsulphate sulphohydrolase, EC
3.1.6.1) and glucose 6-phosphatase (n-glucose
6-phosphate phosphohydrolase, EC 3.1.3.9) were
determined by the methods of Dodgson, Spencer &
Thomas (1955) and Hers, Beaufays & de Duve (1953)
respectively. Inorganic phosphorus was measured
by the method of Allen (1940). RNA polymerase
(RNA nucleotidyltransferase, EC 2.7.7.6) was
assayed by the method of Widnell & Tata (1966).
The transulphuration enzymes were assayed by
the methods of Mudd, Finkelstein, Irreverre & Laster
(1965) with minor modifications.
Methionine adenosyltransferase (ATP: t-methionine-S-adenosyltransferase, EC 2.5.1.6) was assayed
in a mixture (250 /ll total volume) containing: TrisHCI buffer (pH 7,2), 37·5 /lmol; KCI, 50 /lmol;
MgCh, 37·5 /lmol; ATP (made freshly and adjusted
to pH 7'5 with KOH), 5 /lmol; [14C]methionine, 0·5
/lmol; enzyme' (50 /ll; 0·1....Q·5 mg of protein). This
mixture was incubated for 150 min at 37°C. The
reaction was stopped by adding 50 /ll of HCI0 4 (1'0
moljl), the perchlorate precipitated by adding an
equal volume of KOH (1·8 mol/l) and the samples
were cooled to O°C. The precipitated protein and
KCI0 4 were removed by centrifugation at 4°C. The
product, S-adenosylmethionine, was separated from
methionine by descending chromatography on
Whatman no. 1 paper in solvent I (see below). The
chromatograms were dried and 3 ern-wide strips
were cut from the origin to 10 ern along the paper
and the radioactivity was counted. There were no
additional radioactive areas on the chromatogram,
indicating that the reaction product did not decompose during the chromatography.
Cystathionine p-synthase [t-serine hydro-lyase
(adding homocysteine), EC 4.2.1.22] was assayed in
a mixture (350 /ll total volume) containing: Tris-HCI
buffer (pH 8,3), 60 /lmol; EDTA, 1 /lmol; pyridoxal
phosphate, 62·5 /lmol; DL-homocysteine, 10 /lmol;
L-[3- 14C]serine, 0·5 /lmol; enzyme (50 /ll, 0·1....Q·5 mg
of protein). This was incubated for 60 min at 37°C.
The reaction was stopped by adding 100 /ll of HCI
(3·0 mol/I) and perchlorate precipitated by adding
100/l1 of KOH (5-4 moljl); the precipitated protein
and KCI0 4 were separated by centrifugation and the
cystathionine was separated from the serine by
descending chromatography of 20 /ll samples on
Whatman no. 4 paper in solvent II (see below).
Chromatograms were dried and compounds
visualized with ninhydrin (5,6 mmol/I) in acetone;
Transu/phuration enzymes
the orange-brown cystathionine spot was cut out
and the radioactivity counted. Preliminary experiments showed that ninhydrin treatment did not
cause loss of 1 4 C. It was necessary to visualize the
spots because there was a labelled unknown compound running just ahead of the cystathionine; this
contained a constant amount of radioactivity and
was not an impurity in the radioactive substrates
used.
Cystathionine )I-lyase [t-cystathionine cysteinelyase (deaminating), EC 4.4.1.1] was assayed in a
mixture (250 pI total volume) containing: Tris-HCl
buffer (pH 8,4), 50 pmol; pyridoxal phosphate, 125
pmol; cystathionine, 2 pmol; enzyme (50 pI, 0-2-1·0
mg of protein). This was incubated for 20 min at
37°C. The reaction was stopped by adding 250 pI of
500
400
lu
ice-cold dithiothreitol (10 mmol/l) and placing in an
ice bath. The amount of cysteine formed was estimated immediately with an acidic ninhydrin reagent
as described by Gaitonde (1967).
Chromatography was carried out by the descending method, with an overnight run, the following solvents being used: I, methanol-water-pyridine
(20: 5: 1, by vol.); II, propan-1-o1-formic acid-water
(80:6:20, by vol.).
For radioactivity counting the relevant areas of the
chromatograms were immersed in a vial containing
a toluene solution of 2,5-bis-(5-t-butylbenzoxazol2-yl)thiophen (12 mmol/l) and the radioactivity
was measured in a liquid-scintillation spectrometer
(Nuclear-Chicago mark I); 104 counts were collected
on each occasion.
t Loctate
dehydrogenase
t
300
500
-E
§
~
:~
200
400
t Methionire oderosyltrcrsferose t
300
n
200 f-
'0
100 f-
.~
100
Top
Bottom
Top
Bottom
511
~
0
0
500
3000
400
"E
::> 2000
U>
lu
~
200
n0
100
'"
E
>-
0
.s
N
c
w
Cytochrome oxidase
300
c;. 400
.3-
:~
1000
0
500
0::
40
Arylsulphatase
0'
3c
'"
u
~e-,
100
10
u
0
c;.
I-50
.3-
30
Glucose phosphatase
60
>-
U>
::>
U>
0
s:
a.
Cystathionine y -lyase
20
0
"'"
.r
30
40
(;
c.
Cystathionine ,a-synthase
0
0
~
n
.~
'0
300
<5
.s:
M 200
e
8
z- 1500
'in
20
c
'"
Cl
10
0
2
4
6
8
10 12 14 16
Fraction no.
'~
..........-.-....
......
2
4
6
8
10 12 14 16
~
40 -,
~
20
0
*
Fraction no.
FIG. I. Separation of liver subcellular particles by centrifugation on a continuous sucrose gradient. The marker
enzymes identifying the position of the different subcellular components of the liver tissue are as follows: lactate
dehydrogenase, cytosol; cytochrome c oxidase, mitochondria; arylsulphatase, Iysosomes; glucose 6-phosphatase,
endoplasmic reticulum. The enzyme activities are expressed as the stated units per unit of time.
512
Jennifer Allsop and R. W. E. Watts
Results
The results of the density gradient centrifugation
(Fig. 1) show clearly that the activities of methionine
adenosyltransferase, cystathionine p-synthase and
cystathionine y-Iyase all have the same distribution
on the gradient as the cytosol marker enzyme
lactate dehydrogenase. No activity for any of these
enzymes was found in liver cell nuclei, polymorphs,
lymphocytes (with admixed monocytes) or mixed
bone marrow white cells. The enzymes were assayed
on freshly separated cells and subcellular fractions in
order to avoid loss of enzyme on storage (Gaull,
Rassin & Sturman, 1969). The preparations were
disrupted by ultrasonic vibrations (10 s at 8 jlm peak
amplitude, 20 KHz) so that the absence of enzyme
activity is not attributable to enzyme-latency.
Discussion
Mudd et al. (1965) found methionine adenosyltransferase activity in all of the wide range of tissues which
they studied except skeletal muscle, but the distribution of cystathionine p-synthase and cystathionine
y-Iyase was more restricted. They did not, however,
study the subcellular distribution of the enzymes.
Baar & Bickel (1952) made an extensive histopathological study of cystinosis and found cystine
crystals only in the reticuloendothelial cells, the
parenchymal cells of all the organs being unaffected.
The plasma cystine is normal in cystinosis (Crawhall,
Lietman, Schneider & Seegmiller, 1968; Schneider,
Wong, Bradley & Seegmiller, 1968), and abnormally
large amounts of chemically measured cystine have
been found in the Iysosomes of polymorphs, macrophages and cultured skin fibroblasts. However,
parenchymal cells from cystinotic patients have not
been analysed for their cystine content, and the
possibility that they also contain increased amounts
of non-crystalline cystine cannot be completely
excluded.
In the present work the subcellular distribution of
the transulphuration enzymes was established with
liver tissue as a guide to their likely distribution in
other cells, because well-characterized organelle
preparations can be readily obtained from this
material. We found no activity for any of the three
enzymes of the transulphuration pathway in polymorphs, lymphocytes (with admixed monocytes) or
mixed bone marrow white cells. Similarly, Mudd
et al. (1965) and Manowitz, Racevskis & Gilmour
(1970) found no cystathionine p-synthase activity in
mixed peripheral blood leucocytes. Thus the present
findings are incompatible with cystine being formed
in the cytosol of these cells, and then segregated into
modified Iysosomes (or cytosegrosomes) by autophagy (Ericsson, 1969), as suggested by the present
results on liver tissue.
We suggest that, in cystinosis, the affected cells
either acquire their abnormal load of cystine directly
or indirectly from other cells ill vivo, and from the
culture medium ill vitro, or are unable to mobilize
cystine, which has been released by proteolytic
action within their Iysosomes.
References
ALLEN, R.J.L. (1940) The estimation of phosphorus. Biochemical Journal, 34, 858-865.
ApPELMANS, F., W ATIIAUX, R. & DE DUVE, R. (1955) Tissue
fractionation studies. 5. The association ofacid phosphatase
with a special class of cytoplasmic granules in rat liver.
Biochemical Journal, 59, 438-445.
BAAR, H.S. & BICKEL, H. (1952) Morbid anatomy, histology
and pathogenesis of Lignac-Fanconi disease. Acta
Paediatrica 42 (Suppl. 90),171-237.
BLOBELL, G. & POTIER, V.R. (1966) Nuclei from rat liver:
isolation method that combines purity with high yield.
Science, 154, 1662-1665.
COOPERSTEIN, S.J. & LAZAROW, A. (1957) A microspectrophotometric method for the determination of cytochrome
oxidase. Journal of Biological Chemistry, 189, 665-670.
CRAWHALL, J.e. & DAVIES, M.G. (1969) The reduction of
cystine during its transport into rat jejunal everted segments and sacs. Biochemical Journal, 112,571-578.
CRAWHALL, r.c., LIETMAN, P.S., SCHNEIDER, J.A. &
SEEGMILLER, J.E. (1968) Cystinosis. Plasma cystine and
cysteine concentrations and the effect of D-peniciIlamine
and dietary treatment. American Journal of Medicine, 44,
330-339.
CRAWHALL, J.e. & SEGAL, S. (1967) The intracellular ratio
of cysteine and cystine in various tissues. Biochemical
Journal, 105, 891-896.
DANPURE, e.J. (1971) The subcellular distribution of radioactive heavy metals in the rat liver and their effects on
lysosomal enzymes. Ph.D. thesis, University of London.
DODGSON, K.S., SPENCER, B. & THOMAS, J. (1955) Studies on
sulphatases. 9. The arylsulphatases of mammalian livers.
Biochemical Journal, 59, 29-37.
ERICSSON, J.L.E. (1969) Mechanisms of cellular autophagy.
In: Lysosomes in Biology and Pathology, vol. 2, pp. 345-394.
Ed Dingle, J.T. & Fell, H.B. North-Holland Publishing
Co., Amsterdam and London.
GAITONDE, M.K. (1967) A spectrophotometric method for
the direct determination of cysteine in the presence of
other naturally occurring amino acids. Biochemical Journal,
104, 627-633.
GAULL, G.E., RASSIN, D.D. & STURMAN, J.A. (1969) Enzymatic and metabolic studies of homocystinuria. Neuropiidiatrie, 1, 199-226.
HERS, H.G., BEAUFAYS, H. & DE DUVE, C. (1953) L'analyse
simultanee des hexoses, des trioses et de leurs esters
phosphores. Biochimica et Biophysica Acta, 11, 416-426.
Transulphuration enzymes
HUMMELER, K., ZAJAC, B.A., GENEL, M., HOLTZAPPLE, P.G.
& SEGAL, S. (1970) Human cystinosis: intracellular
deposition of cystine. Science, 168, 859-860.
MANOWJTZ, P., RECEVSKIS, J. & GILMOUR, D.G. (1970) A
modified assay for cystathionine synthase. C/inica Chimica
Acta, 28, 269-276.
MUDD,S.H., FINKELSTEIN, J.D., IRREVERRE, F. & LASTER, L.
(1965) Transulphuration in mammals. Microassays and
tissue distribution of three enzymes of the pathway. Journal
of Biological Chemistry, 240, 4382-4392.
PLUMMER, D.T. & WJLKINSON, J.H. (1963) Organ specificity
of lactate dehydrogenase activity. 2. Some properties of
human-heart and liver preparations. Biochemical Journal,
87,423-429.
RAHMAN, Y.E. & LINDENBAUM, A. (1964) Lysosome particles
and subcellular distribution of polymeric tetravalent
plutonium 239. Radiation Research, 21, 575-583.
SCHNEIDER, J.A., BRADLEY, K.H. & SEEGMILLER, J.E. (1968)
D
513
Transport and intracellular fate of cysteine- 3sS in leukocytes from normal subjects and patients with cystinosis.
Paediatric Research, 2, 441-450.
SCHNEIDER, J.A., WONG, V., BRADLEY, K. & SEEGMILLER, J.E.
(1968) Biochemical comparisons of the adult and childhood
forms of cystinosis. New England Journal of Medicine, 279,
1253-1257.
WESTWJCK, W.J., ALLSOP, J. & WATTS, R.W.E. (1972) A
study of the effect of some drugs which cause agranulocytosis on the biosynthesis of pyrimidines in human
granulocytes. Biochemical Pharmacology, 21, 1955-1966.
WIDNELL, C.C. & TATA, J.R. (1966) Studies on the stimulation by ammonium sulphate of the DNA-dependent
RNA polymerase of isolated rat-liver nuclei. Biochimica et
Biophysica Acta, 123, 478-492.
WILLCOX, P. & PATRICK, A.D. (1974) Biochemical diagnosis
of cystinosis using cultured cells. Archives of Disease in
Childhood, 49, 209-212.