Journal of Nriirocht.r~~isrry. 1975. Vol. 25. pp. 871 876. Pergamon Picas. Printed in Great Britain.
THE BINDING OF TRIETHYLTIN TO RAT BRAIN MYELIN E. A. LOCK’and W. N. ALtmDGE Biochemical Mechanism Section, Toxicology Unit, Medical Research Council Laboratories, Woodmanstcrne Road, Carshalton, Surrey, U.K.
(Received 17 March 1975. Accepted 8 M a y 1975) Abstract-A high affinity binding site for triethyltin was found in rat brain myelin with an affinity of approx 6.6 x 1 0 ’ ~ ~at ’pH 7.5. Competitive binding studies showed that tricthyl-lcad had about the same affinity and trimethyltin 30 times lower affinity than triethyltin. Hexachlorophane and 3,sdiiodo-4-chlorosalicylanilide did not prevent tricthyltin binding to rat brain myelin. Since triethyltin, hexachlorophane and 3,5-diiodo-4-chlorosalicylanilideall produce similar oedematous lesions in the brain of rats, whereas triethyl-lead and trimethyltin do not, it is concluded that the high affinity triethyltin binding site either is not involved or is not the only factor in oedema production.
TRIETHYLTIN produces an oedematous lesion in the white matter of the central nervous system of rats (MAGEEet al., 1957). This lesion when examined by electron microscopy shows accumulation of fluid between and splitting of thc myelin layers at the intraperiod line (ALEU et al., 1963; LEE& BAKAY,1965). More recently it has been reported that triethyltin given to rats over long periods causes demyelination in the brain (ETOet al., 1971 ; Smm, 1973). High concentrations of triethyltin are found in the blood and liver of rats after injection of the compound (ROSE& ALDRIDGE, 1968). In the guinea-pig, the liver was the organ containing the highest concentration of triethyltin (ROSE& ALDRIDGE,1968). Triethyltin present in rat blood was shown to be in the erythrocytes and due to the binding of triethyltin to rat haemoglobin (ROSE& ALDRIDGE, 1968). Similarly, 70% of the triethyltin in guinea-pig liver was shown to be bound to a soluble protein present in the cytoplasm (ROSE& LOCK,1970). Based on photooxidation studies and the known chemistry of triethyltin, evidence has been presented that the triethyltin binding sites in both rat haemoglobin and guineapig liver protein consist of paired histidine residues (ROSE, 1969; ROSE& LOCK,1970). The mechanism of production of cerebral oedema by triethyltin is not known. After administration of triethyltin chloride to rats, isolation of subcellular fractions of rat brain indicated that triethyltin was present in fractions which would be expected to contain myelin (ROSE& ALDRIDGE, 1968). Since the lesion appears to involve splitting of myelin, the ability of myelin to bind tricthyltin was examined. Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilide produce an oedematous lesion in the central nervous system Present address: Imperial Chemical Industries Limited. Central Toxicology Laboratory, Alderley Park, Nr Macclesfield, Cheshirc. SKI0 4TJ, U.K.
similar to triethyltin (KIMBROUGH & GAINS,1971; KURTZet al., 1969; LOCK,1974) whereas trimethyltin and triethyl-lead do not (BAKSES& STONER,1958; CREMER,1959). The ability of these compounds to compete with triethyltin for binding sites in myelin was also examined. METHODS AND MATERIALS Special chemicals Triethyl[113Sn]tin chloride was purchased from the Radiochemical Centre, Amersham, Bucks, U.K. The specific radioactivity wdS 6 mCi/mmol. Stock 0.2 M solution in ethanol was diluted in water to give 2mM solutions for binding studies from which further dilutions were made. The following gifts are acknowledged: hexachlorophane [2,2’-methylenebis-(3,4,6-trichlorophenol)] from the Cooper Technical Bureau, Berkhamsted, Herts, U.K., trimethyltin acetate and triethyl-lead acetate from Dr G. J. M. Van der Kerk, and Clioxanide (2-acetoxy-3,5-diiodo-4chlorobenzanilide) from Parke, Davis Co., Detroit, Michigan, U.S.A. Clioxanide was converted to 3.5-diiodo-4chlorosalicylanilide by alkaline hydrolysis.
Isolation of myelin Myelin was prepared from rat brain based on the procedure of NORTON(1971). Male rats (200g body wt) of the Porton strain were used. Two whole brains were homogenized in 0.32 M-sucrose by using a homogenizer with a total clearance of 0.25 mm and a speed of rotation of 1950 rev./min (ALDRIDGE ef ul., 1960; WEBSTER & SMITH,1964). The homogenate was diluted to 5%, w/v with ice-cold 0.32 M-sucrose, then laycred over 085 M-sucrose and centrifuged at 75,000g for 30min at 0°C. The myelin layer at the interphase was removed, dispersed in distilled water and re-centrifuged at 75,OOOg for 15 min. The pellet collected at this stage was called the ‘crude myelin fraction’. For some experiments the myelin was purified further: the pellet was dispersed in ice-cold water and centrifuged at 12,000g for 10min at 0°C; this step was repeated. The pellet obtained was then resuspended in ice-cold 0.32 Msucrose, layercd over ice-cold 0.85 M-sucrose and centrifuged at 75,000 g for 30 min at 0°C. The myelin layer at
871
E. A. LOCKand W. N. ALDRIDGE
872
the interphase was collected, diluted with water, centrifuged at 12,000g for 10 min. The pellet obtained was called the ‘purified myelin fraction’. The myelin fractions were resuspended in 10-25 ml ice-cold 0.32 M-sucrose and used immediately. Binding of triethyltin to rat brain myelin fractions Binding of triethylc’ ‘3Sn]tin chloride to rat brain myelin was measured as follows: rat brain myelin (1 ml equivalent to 2-4mg protein) was incubated at 20°C for 15 min in polypropylene tubes in 100 mM-Tris-chloride buffer, pH 7.5 (4 ml) containing the triethyll’ ‘’Snltin chloride. After incubation the tubes were centrifuged at 40,000g at 20°C for 30min. The supernatant and pellet were separated and the tube drained and wiped free from supernatant. The radioactivities of the whole of the pellet (resuspended in dilute sodium deoxycholate) and 4ml of supernatant were measured and the content of triethyltin calculated by reference to a sealed radioactive standard solution of known concentration. The results were plotted by the procedure of SCATCHARD (1949) and analysed by trial and error.
phosphohydrolase by the method of OLAFSON et al. (1969) with the following two modifications: the concentration of buffer was increased to 0.1 M to provide additional buffering capacity, and sodium deoxycholate (0.2%)was added to maximally activate the enzyme. The inorganic phosphate liberated by this technique was determined by the method of BERENBLUM & CHAIN(1938) as modified by THRELFALL (1957). RESULTS
Characterization of rat brain myelin ,$-actions When the purified myelin fraction was examined under the electron microscope it had a typical myelinic membranous structure and was virtually free of recognizable fragments of other cellular organelles or membranes. Occasionally a few mitochondria could be seen trapped in the myelin vesicles. Twenty-five per cent of the protein in whole brain was found in the crude myelin fraction (Table 1) and this was reduced upon purification to 12% of the total. About 50% of the myelin marker enzyme 2’-3’ Measurement of radioactiuity cyclic nucleotide-3’-phosphohydrolase (KURIIIARA & Triethyltin chloride labelled with “’Sn, a gamma emit- TSUKADA, 1967; OLAFSON et al., 1969) was found in ting isotope, was counted with.an overall efficiency of 9% the crude myelin fraction (Table 1) and this was in a Panax Gamma Counter. For details of the decay pro- reduced upon purification to 21% of the whole brain cess see ROSE& ALDRIDGE (1968). activity. The specific activity of the 2’-3’cyclic nucleotide-3’-phosphohydrolase present in the crude Electron microscopy and purified myelin was the same (6pmoI P, liberSamples were fixed in buffered 2% osmium tetroxide ated/min/mg of protein), indicating that the procedure solution pH 7.5, then washed several times in buffer. Specimens were stained in uranyl acetate and then lead citrate. had removed protein and enzyme activity together. Lactate dehydrogenase contamination of the crude Analytical methods myelin was very small (2%) and on purification this The following methods were used: protein was deter- activity was completely removed. Cytochrome oximined by the biuret method of GORNALLet al. (1949), in- dase, a specific marker for mitochondria, showed corporating the modification of HES & LEWIN(1965) for some activity in the crude myelin fraction (4%).The proteolipid proteins. Sodium deoxycholate was added to purified myelin still had some activity, although very solubilie all fractions. Bovine serum albumin was used small, and this is consistent with the electron microas a standard; lactate dehydrogenase(L-1actate:NAD’ oxidoreductase, EC 1.1.1.27) was assayed as described by scopy studies. The most likely contaminant of the BERGMEYER (1963); cytochrome c oxidase (EC 1.9.3.1) by myelin fraction is other membranous material. To the method described by SCHNAITMAN et al. (1967); acetyl- detect this, the enzyme marker acetylcholinesterase cholinesterase (acetylcholine hydrolase, EC 3. I. 1.7) as de- was used. The radiochemical method used in this scribed by SIAKOTOS et al. (1969); 2’-3’ cyclic nucleotide-3’- study is very sensitive and 14% of the total activity TABLE1. PURITYOF Protein content or marker enzyme activities
Protein (mg/g wet wt) Lactate dehydrogenase (pmol NADH/min/g wet wt) Cytochrome oxidase (pg atom O/min/g wet wt) Acetylcholinesterase (pmol acetylcholine hydrolysed/ min/g wet wt) 2’-3’cyclic nucleotide 3‘-phosphohydrolase (pmol P,/min/g wet wt)
RAT BRAIN MYELIN FRACTIONS
Whole homogenate
Crude myelin fraction
+ 1.3
33.4 f 1.8 (9)
15.7 f 1-0 (10)
47.3 f 2.8 (4)
0 8 8 f 0.16 (4)
0
46.4 f 3.2 (5)
1.72 f 0.23 (4)
0.1 1 f 0.03 (5)
9.09 f 025(7)
1.24 & 0.04 (5)
0.06 f 0.01 (5)
+ 14
84.5 f 9.0 (6)
130.3
4 0 4 0 k 17
(23)
(5)
196.0
(5)
Purified myelin fraction
(4)
The figures in the table represent the protein content and enzyme activities found in the whole brain homogenate and myelin fractions derived from 1 g wet weight of brain. The enzyme and protein analysis was carried out as described in the text. Values are expressed as mean f S.E.M. with the number of observations in brackets.
Binding of triethyltin to myelin
o;I
1:
The equation for competitive binding of A at a single site in the presencc of a competing molecule B is v=
O.'
873
I I
2
3
4
+
nKArA1 KACAI
FIG.1. Binding of triethyltin to purified rat brain myelin. Thc solid line is that calculated for a 2 component system of binding sites 1 and 2 with thc following constants: n,, 0.54nmol/mg ofprotein; K I , 6.6 x lo5 M-'; n,, 13.5 nmol/ mg of protein; K,, 2.6 x 104M-'.
EDSALL& WYMAN (1958)
where V is the concentration of bound ligand/mg of protein, n is the concentration of total binding sites/ mg of protein, A is the concentration of free ligand and B is the concentration of free competing ligand. K A is the affinity constant for the binding of A and K, the affinity constant for the binding of B. With a range of concentrations of A and a fixed concentration of competitor B, where K' is the apparent affinity constant measured in the presence of B and the binding of A is measured V -
A
5
7(nrnol of triethyltin /rng of protein)
+ KBCB1
= K'(n
- vA).
Plotting VA/A against VA gives a line with a slope - K' and intercept of nK' on the y-axis and n on the x-axis (Fig. 2). The affinity constant K , may then be calculated from
K,
K, - K'
= ___
K"B1
When [B]/[A] is a constant (z), was found in the crude myelin but only 0.6% in the purified fraction. Having obtained a myelin fraction from rat brain which was free from major contaminating particles, the binding of triethyltin to this fraction was examined. Binding of trielhyltin to rut brain myelin A Scatchard analysis of the data for the binding of triethyl[113Sn]tin to purified myelin gave a curve (Fig. 1) indicating more than one class of binding sites (EDSALL& WYMAN, 1958; SCATCHARD, 1949). A satisfactory fit to the results may be obtained by a minimum of 2 classes of sites having constants of the value n , , 054 f 0.03 nmol/mg of protein; K , , 6.6 f 0.7 x 1 0 ' ~ ~ 'n2, ; 133 f 2.3 nmol/mg of protein; K,, 2.6 & 0.5 x lo4 M-' (mean of 7 experiments). The mean values of the constants obtained from 8 experiments using the crude myelin fraction were as follows: n,, 050 i 0.03 nmol/mg of protein; K , , 6.6 0.3 x lo5 M-'; nz, 14.1 f 1.1 nmol/mg of protein; K,, 2 3 f 0.1 x lo4 M-'. These results are identical with thosc found for the purified myelin and therefore for convenience the crude myelin fraction was used for subsequent experiments. Influence of other substances on the binding of triethyltin to rat brain myelin A number of other compounds were examined as competitors for the binding of triethyltin to the crude myelin fraction, and their binding constants determined from the results of these experiments.
V -
A
= nK, -
V,(KA
+ K, . z).
Then plotting P,JA against VA gives a line with slope - ( K A + K , .z) and an intercept of nKAon the y-axis and [n/(l K , .z/KA)] = n' on the x-axis (Fig. 2). From the experimentally determined values of the
+
nK
nK
Q, lh CCornpetitor I f ixed
n'
n
Y FIG.2. Diagrammatic representation of competitive binding at a single site (a) using a fixed concentration of competitor; where the number of binding sites is unaltered but the affinity constant is lowered; (b) using a constant ratio of competitor to triethyltin; where the number of binding sites is reduced and the affinity constant increases.
E. A. LOCK and W. N. ALDRIDGE
874
slope and total bound ligand A(n') the affinity constants K B for the competitor B may be calculated. Scatchard plots of experimental results of the binding of triethyl[113Sn]tin to rat brain myelin in the presence of 9.4 PM triethyl-lead or in the presence of a range of concentrations of triethyl-lead such that the ratio of triethyL1ead:triethyltin is 1.0 are shown in Figs. 3 and 4 respectively. They show examples of the general principles outlined above for the two experimental situations (cf Fig. 2) but for the more complicated situation where both triethyltin and triethyl-lead bind to two sites 1 and 2. When analysing a two-component system by curve fitting, the accuracy of the procedure is greatly influenced by the relative sues of n,, n2, K and K,. With the method using a constant concentration of competitor it becomes more difficult to separate a two-component system when the relative affinity constants are one order or less apart. For this reason the method using the constant ratio of competitor where the affinity constants increase is considered more accurate. The values for K , . measured in the presence of competitor, have been used to calculate KB. The binding of triethyl["3Sn]tin to rat brain myelin in the presence of hexachlorophane, 3,5-diiodo-4chlorosalicylanilide, trimethyltin and triethyl-lead was measured and the derived constants are tabulated in Table 2. Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilide had no observable effect on triethyltin binding to rat brain myelin. Trimethyltin competes with triethyltin for the high affinity binding site (1) in mye0.8
07
x
3.
=
\
c .m
c
05
2 R
r 0
-E
s
I I
I
I
2
3
I 4
I 5
B(nrnolof rriethyltin /rng ofprotein)
FIG.4. The effect of triethyl-lead on the binding of triethyltin to rat brain myelin. The solid lines are the best fit; analysis gave the following constants: Control (0) n,, 0,535nmol/mg of protein; KL.6 x lo5 M-'; n,, 10-4nmoli mg of protein; K , , 2 3 x 1 0 4 K 1 and triethyl-lead was present so that the [triethyl-lead]/[triethyltin] ratio was I : ] (0)n,, 042 nmol/mg of protein; K , . 9 x lo5 M - l ; n2. 6.67 nmol/mg of protein; K 2 , 3 x lo4 M -
lin. At a fixed concentration of trimethyltin the affinity for triethyltin is progressively lowered. whereas at a constant ratio of trimethyltin to triethyltin the affinity for triethyltin is increased (Table 2). An affinity constant of 2.1 f 0.5 x lo4 M - ' for trimethyltin binding to the high affinity site was calculated. Triethyl-lead also competes with triethyltin for the high affinity binding site (1) for myelin. At a fixed concentration of triethyl-lead the affinity for triethyltin is lowered, whereas at a constant ratio of triethyllead to triethyltin a progressive increase in affinity and decrease in number of triethyltin binding sites is observed (Table 2). The derived affinity constant for triethyl-lead was 2.7 f 0.5 x lo5 M - and is very similar to the affinity constant for triethyltin. DISCUSSION
0.4
k-l
a -
I
03
0.2
I
I
I
I
2
3
I 4
1 5
F ( n m o l of triethyltin /mg of protein) FIG. 3. The effect of triethyl-lead on the binding of triethyltin to rat brain myelin. The solid lines are the best fit; analysis gave the following constants: Control (0) n,, O-92nmol/mg of protein; K,, 5 x lo5 M - ' ; n,, 12.4nmoI/ mg of protein, K 2 , 2.5 x l O 4 K 1 and for triethyl-lead 9.4 pM (O), n,, 1.3 nmol/mg of protein, K,, 1 x lo5 M-'; n2. 20nmol/mg of protein; K , , 1 x 104hl-'.
Myelin was separated from rat brain and shown to be free from major contamination. Purified rat brain myelin binds triethyltin. The results when analysed by the method of SCATCHARD (1949) may be interpreted by a minimum of 2 classes of sites. One class of binding sites has an affinity of about 6.6 x lo5 M-' and a concentration of 0.54 nmol/mg of protein. The other class of site is of much lower affinity and higher concentration (2.6 x lo4 M - ' and 13.5 nmol/mg protein). Therefore triethyltin binds to rat brain myelin with an affinity similar to its affinity for rat haemoglobin (ROSE, 1969), for rat liver mitochondrial binding site (1) (ALDRIDGE& STREET,1970) and for a protein from guinea-pig liver supernatant fraction (ROSE & LOCK, 1970) (cf Table 3 for summary. The binding of triethyltin to rat haemoglobin and
Binding of triethyltin to myelin TABLE2. THEEFFECT OF
VARIOUS COMPOUNDS ON TH~.BINDINGOF TRIETHYLTIN TO RAT BRAIN MYELIN
"1
(nmol/mg of protein)
Compound Triethyltin (8) Trimethyltin
94pM 18-8pM
Trimeth Itin Triethitin
[
]
0.50 0.84 1.11
Triethyl-lead
K,
W1) 6.6
0.03
0.38
7'5
30
0 3 x lo5 5.0 105 3.0 105 _+
9.0
n2 (nmol/mg of protein)
(M-7
14.1 & 1.1 20.0 124
2.5 _+ 0.1 x lo4 2.0 x 104 2.5 x 104
105
9.8
10.0 x 10' 1.0 105
11.7 20.0
K2
KB
(M-
l)
2.1 x 104 3.5 x 104
3.5 x 104 3.0 x 104 1.0 104
1.3 x 104 4.2 x 105
1.3 x 104
94pM
0.55 1-30
1.0(2)
0.40
9.0 x lo5
3.0 x 105
0.33 0.08
18.0 x 105 80.0 x lo5
6.9 4.3 7.0
3.0 x lo4
1.5
3.0 x 104 1.0 104
1.6 x 105 1.5 x 105
9.7
3.0
50
[Hexachlorophane Triethyltin ] ''O 10.0
0.55
6.0 x 105
0.41
7.0
I ,o
0.44
[Salicylanilide] Triethyltin
875
104
105
3.0 x 104
For the preparation of myelin and the determination of binding see the Materials and Methods scction. The crude myelin fraction was used for these experiments. The results are single observations cxcept for triethyltin where they are expressed as mean f S.E.M.(No. of observations) n, and n2 are the concentration of binding sites I and 2; and K 1 and K , are their aRnity constants. K , is the derived affinity constant for the competitor at the high affinity binding site. It was calculated by knowing the K , for triethyltin and the K' for triethyltin in the presence of the competitor, see EDSALL& WYMAN(1958). guinea-pig liver supernatant fraction is sensitive to photo-oxidation, and the loss of binding was correlated with the loss of 2 histidine residues per binding site (ROSE, 1969; ROSE & LOCK, 1970). This finding is supported by the known chemistry of these compounds (POLLER, 1965; LUITJEN et al., 1962). In a single experiment, photo-oxidation of rat brain myelin fraction in the presence of methylene blue for 20min greatly reduced the number of high affinity triethyltin binding sites. On the basis of an analogy between the binding of triethyltin to other proteins, it is suggested that it binds to the protein moiety in myelin, and because of its sensitivity to photo-oxidation may 1961; WEILet involve histidine (RAY & KOSHLAND, a/.: 1951). The isolation and separation of 3 main types of protein from rat brain myelin has been fairIy well characterized (MARTENSON & GAITONDE, 1969; MEHL& HALARIS, 1970)and future study could establish whether triethyltin binds to 1 of these main protein classes. Triethyl-lead and trimethyltin compete with triTAULE 3. AFFINITY CONSTANTS FOR
Macromolecule Rat haemoglobin (ROSE,1968, 1969) Rat liver mitochondria (ALDRIDGE&
STREET,
THE BINDING OF TRIETHYLTIN IKIMETHYLTIN A N D TRIETHYL-LEAD TO MACROMOLECULES
Triethyltin
Affinity constant trimethyltin
3.5 x 1 0 5 ~ - 1
2.8
4.7 x 105 M - '
1.2
X
Triethyl-lead
1OsM-'
-
104~-1
-
-
-
1970)
Protein fraction from g. pig liver supernatant (ROSE& LOCK,1970) Rat brain myelin
2.0
X
106M-'
6.6 x 1 0 5 ~ - '
* Measured indirectly by competition with triethyltin. Not known.
ethyltin for the high affinity binding site in myelin. Triethyl-lead has a derived affinity constant for rat brain myelin of 2.7 x lo5 M - I (Table 3), very similar to that of triethyltin whereas trimethyltin has a thirtyfold lower affinity constant than triethyltin at 2.1 x lo4 M-' (Table 3). The affinity constant for the binding of trimethyltin to rat liver mitochondria is also about thirty times lower than that for triethyltin (ALDRIDCE & STREET,1970) (Table 3). Neither triethyl-lead nor trimethyltin produces an oedematous lesion in the central nervous system (BARNES& STONER, 1958; CREMER, 1959). Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilided o not compete with tricthyltin for the high affinity binding site in myelin. However, both these compounds produce an oedematous lesion in the central nervous system similar to triethyltin (KIMBROUGH & GAINES,1971 ; KURTZet al., 1969; LOCK, 1974). Therefore the high affinity binding to rat brain myelin either is not involved or is not the only factor involved in the production of oedema by these compounds.
2.1
x
1 o 4 ~ - I *
2.7 x 105 M-
'*
876
E. A. LOCKand W. N. ALDRIDGE
LQCKE. A. (1974) Ph.D. 7hesis, CNAA. LUUTENJ. G. A.: JANSSENM. J. & VAN DER KERKG. J. M. (1962) Reel Trav. chim. Pay-Bas Belg. 81. 202-205. REFERENCES MACFEP. N., STONERH. B. & BARNESJ. M. (1957) J. path. Bact. 73. 107-124. B. W. (1970) Biochem. J . 118. ALDRIDGE W. N. & STREET MEHLE. & HALARJS A. (1970) J. Neurochem. 17. 659-668. 171-1 79. R. E. & GAITONDE M. K. (1969) J. Neurochem. ALDRIDGEW. N., EMERYR. C. & STREETB. W. (1960) MARTENSON 16. 889-898. Biochem. J. 77, 326-327. ALEUF. P., KATZMAN R. & TERRY R. D. (1963) J . Neuro- NORTONW. T . (1971) Adv. exp. med. B i d . 13. 327--337. OLAFSON R. W., DRUMMOND G. 1. & LEE J. F. (1969) Can. path. exp. Neurol. 22. 403413. J . Biochem. 47. 961-966. BARNESJ. M. & STONER H. B. (1958) Br. J. industr. Med. 15. 1 s 2 2 . POLLER R. C. (1965) J . Organornet. Chear. 3. 321-329. RAYW. J. & KOSHLANDD. E. (1961) J . bid. Chem. 236. BERENBLUM I. & CHAINE. (1938) Biochem. J . 32. 286-298. BERGMEYER 1973-1 979. H. (1963) Methods of Enzymatic Analysis. Academic Press, New York. ROSE M. S . (1968) Ph.D. Thesis, University of London. CREMER J . E. (1959) Br. J . industr. Med. 16. 191-199. ROSEM. S. (1969) Biochem J . 111. 129--137. EDSALLJ. T . & WYMANJ. (1958) Biophysical Chemistry, ROSEM. S . & ALDRIDCEW. N. (1968) Biochem. J . 106. Vol. 1, pp. 651-653. Academic Press, New York. 821-828. ETO Y., SUZUKIK. & SuzuK1 K. (1971) J. Lipid Res. 12. ROSEM. S . & LOCKE. A. (1970) Biochem. J. 120. 151-157. 570-579. SCATCHARD G. (1949) Ann. N.Y. Acad. Sci. 51. 660-672 GORNALL A. K., BARDAWILL C. J. & DAVIDM. M. (1949) SCHNAITMAN J. W. (1967) C., ERWINV. G. & GREENAWALT J . bid. Chem. 177. 751-766. J . Cell. B i d . 32. 719-735. HESSH. H. & LEWINE. (1965) J. Neurochem. 12. 205-21 1. SMKOTOSA. N., FILBERT M. & HESTER R. (1969) Biochem. KIMBROUGH R. D. & GAIN&T . B. (1971) Archs enuiron. Med. 3. 1-12. Health 23. 114-118. SMITH M. E. (1973) J. Neurochem. 21. 357-372. KURIHARAT. & TSUKADA Y. (1967) J. Neurochrm. 14. THRELFALL C. J. (1957) Bwchem. J . 65. 694699. 1167-1 174. WEBSTER G. R. & SMITHA. T. (1964) Biochem. J. 90, 64-65. KURTZS.M., SCHARDEIN J. L., FITZGERALD J. E. & KAUMP WEIL L., GORDON W. G. & BUCHERTA. R. (1951) Archs D. H. (1969) Tox. appl. Pharmac. 14. 652. Biochem. Biophys. 33. 90-109. LEE J. C. & BAKAYL. (1965) Archs Neurol. Psychiat. Chicago 13, 48-57. Acknowledgement-The authors thank Dr W. H. BUTLERfor the electron microscopy on the myelin fractions.
THE BINDING OF TRIETHYLTIN TO RAT BRAIN MYELIN E. A. LOCK’and W. N. ALtmDGE Biochemical Mechanism Section, Toxicology Unit, Medical Research Council Laboratories, Woodmanstcrne Road, Carshalton, Surrey, U.K.
(Received 17 March 1975. Accepted 8 M a y 1975) Abstract-A high affinity binding site for triethyltin was found in rat brain myelin with an affinity of approx 6.6 x 1 0 ’ ~ ~at ’pH 7.5. Competitive binding studies showed that tricthyl-lcad had about the same affinity and trimethyltin 30 times lower affinity than triethyltin. Hexachlorophane and 3,sdiiodo-4-chlorosalicylanilide did not prevent tricthyltin binding to rat brain myelin. Since triethyltin, hexachlorophane and 3,5-diiodo-4-chlorosalicylanilideall produce similar oedematous lesions in the brain of rats, whereas triethyl-lead and trimethyltin do not, it is concluded that the high affinity triethyltin binding site either is not involved or is not the only factor in oedema production.
TRIETHYLTIN produces an oedematous lesion in the white matter of the central nervous system of rats (MAGEEet al., 1957). This lesion when examined by electron microscopy shows accumulation of fluid between and splitting of thc myelin layers at the intraperiod line (ALEU et al., 1963; LEE& BAKAY,1965). More recently it has been reported that triethyltin given to rats over long periods causes demyelination in the brain (ETOet al., 1971 ; Smm, 1973). High concentrations of triethyltin are found in the blood and liver of rats after injection of the compound (ROSE& ALDRIDGE, 1968). In the guinea-pig, the liver was the organ containing the highest concentration of triethyltin (ROSE& ALDRIDGE,1968). Triethyltin present in rat blood was shown to be in the erythrocytes and due to the binding of triethyltin to rat haemoglobin (ROSE& ALDRIDGE, 1968). Similarly, 70% of the triethyltin in guinea-pig liver was shown to be bound to a soluble protein present in the cytoplasm (ROSE& LOCK,1970). Based on photooxidation studies and the known chemistry of triethyltin, evidence has been presented that the triethyltin binding sites in both rat haemoglobin and guineapig liver protein consist of paired histidine residues (ROSE, 1969; ROSE& LOCK,1970). The mechanism of production of cerebral oedema by triethyltin is not known. After administration of triethyltin chloride to rats, isolation of subcellular fractions of rat brain indicated that triethyltin was present in fractions which would be expected to contain myelin (ROSE& ALDRIDGE, 1968). Since the lesion appears to involve splitting of myelin, the ability of myelin to bind tricthyltin was examined. Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilide produce an oedematous lesion in the central nervous system Present address: Imperial Chemical Industries Limited. Central Toxicology Laboratory, Alderley Park, Nr Macclesfield, Cheshirc. SKI0 4TJ, U.K.
similar to triethyltin (KIMBROUGH & GAINS,1971; KURTZet al., 1969; LOCK,1974) whereas trimethyltin and triethyl-lead do not (BAKSES& STONER,1958; CREMER,1959). The ability of these compounds to compete with triethyltin for binding sites in myelin was also examined. METHODS AND MATERIALS Special chemicals Triethyl[113Sn]tin chloride was purchased from the Radiochemical Centre, Amersham, Bucks, U.K. The specific radioactivity wdS 6 mCi/mmol. Stock 0.2 M solution in ethanol was diluted in water to give 2mM solutions for binding studies from which further dilutions were made. The following gifts are acknowledged: hexachlorophane [2,2’-methylenebis-(3,4,6-trichlorophenol)] from the Cooper Technical Bureau, Berkhamsted, Herts, U.K., trimethyltin acetate and triethyl-lead acetate from Dr G. J. M. Van der Kerk, and Clioxanide (2-acetoxy-3,5-diiodo-4chlorobenzanilide) from Parke, Davis Co., Detroit, Michigan, U.S.A. Clioxanide was converted to 3.5-diiodo-4chlorosalicylanilide by alkaline hydrolysis.
Isolation of myelin Myelin was prepared from rat brain based on the procedure of NORTON(1971). Male rats (200g body wt) of the Porton strain were used. Two whole brains were homogenized in 0.32 M-sucrose by using a homogenizer with a total clearance of 0.25 mm and a speed of rotation of 1950 rev./min (ALDRIDGE ef ul., 1960; WEBSTER & SMITH,1964). The homogenate was diluted to 5%, w/v with ice-cold 0.32 M-sucrose, then laycred over 085 M-sucrose and centrifuged at 75,000g for 30min at 0°C. The myelin layer at the interphase was removed, dispersed in distilled water and re-centrifuged at 75,OOOg for 15 min. The pellet collected at this stage was called the ‘crude myelin fraction’. For some experiments the myelin was purified further: the pellet was dispersed in ice-cold water and centrifuged at 12,000g for 10min at 0°C; this step was repeated. The pellet obtained was then resuspended in ice-cold 0.32 Msucrose, layercd over ice-cold 0.85 M-sucrose and centrifuged at 75,000 g for 30 min at 0°C. The myelin layer at
871
E. A. LOCKand W. N. ALDRIDGE
872
the interphase was collected, diluted with water, centrifuged at 12,000g for 10 min. The pellet obtained was called the ‘purified myelin fraction’. The myelin fractions were resuspended in 10-25 ml ice-cold 0.32 M-sucrose and used immediately. Binding of triethyltin to rat brain myelin fractions Binding of triethylc’ ‘3Sn]tin chloride to rat brain myelin was measured as follows: rat brain myelin (1 ml equivalent to 2-4mg protein) was incubated at 20°C for 15 min in polypropylene tubes in 100 mM-Tris-chloride buffer, pH 7.5 (4 ml) containing the triethyll’ ‘’Snltin chloride. After incubation the tubes were centrifuged at 40,000g at 20°C for 30min. The supernatant and pellet were separated and the tube drained and wiped free from supernatant. The radioactivities of the whole of the pellet (resuspended in dilute sodium deoxycholate) and 4ml of supernatant were measured and the content of triethyltin calculated by reference to a sealed radioactive standard solution of known concentration. The results were plotted by the procedure of SCATCHARD (1949) and analysed by trial and error.
phosphohydrolase by the method of OLAFSON et al. (1969) with the following two modifications: the concentration of buffer was increased to 0.1 M to provide additional buffering capacity, and sodium deoxycholate (0.2%)was added to maximally activate the enzyme. The inorganic phosphate liberated by this technique was determined by the method of BERENBLUM & CHAIN(1938) as modified by THRELFALL (1957). RESULTS
Characterization of rat brain myelin ,$-actions When the purified myelin fraction was examined under the electron microscope it had a typical myelinic membranous structure and was virtually free of recognizable fragments of other cellular organelles or membranes. Occasionally a few mitochondria could be seen trapped in the myelin vesicles. Twenty-five per cent of the protein in whole brain was found in the crude myelin fraction (Table 1) and this was reduced upon purification to 12% of the total. About 50% of the myelin marker enzyme 2’-3’ Measurement of radioactiuity cyclic nucleotide-3’-phosphohydrolase (KURIIIARA & Triethyltin chloride labelled with “’Sn, a gamma emit- TSUKADA, 1967; OLAFSON et al., 1969) was found in ting isotope, was counted with.an overall efficiency of 9% the crude myelin fraction (Table 1) and this was in a Panax Gamma Counter. For details of the decay pro- reduced upon purification to 21% of the whole brain cess see ROSE& ALDRIDGE (1968). activity. The specific activity of the 2’-3’cyclic nucleotide-3’-phosphohydrolase present in the crude Electron microscopy and purified myelin was the same (6pmoI P, liberSamples were fixed in buffered 2% osmium tetroxide ated/min/mg of protein), indicating that the procedure solution pH 7.5, then washed several times in buffer. Specimens were stained in uranyl acetate and then lead citrate. had removed protein and enzyme activity together. Lactate dehydrogenase contamination of the crude Analytical methods myelin was very small (2%) and on purification this The following methods were used: protein was deter- activity was completely removed. Cytochrome oximined by the biuret method of GORNALLet al. (1949), in- dase, a specific marker for mitochondria, showed corporating the modification of HES & LEWIN(1965) for some activity in the crude myelin fraction (4%).The proteolipid proteins. Sodium deoxycholate was added to purified myelin still had some activity, although very solubilie all fractions. Bovine serum albumin was used small, and this is consistent with the electron microas a standard; lactate dehydrogenase(L-1actate:NAD’ oxidoreductase, EC 1.1.1.27) was assayed as described by scopy studies. The most likely contaminant of the BERGMEYER (1963); cytochrome c oxidase (EC 1.9.3.1) by myelin fraction is other membranous material. To the method described by SCHNAITMAN et al. (1967); acetyl- detect this, the enzyme marker acetylcholinesterase cholinesterase (acetylcholine hydrolase, EC 3. I. 1.7) as de- was used. The radiochemical method used in this scribed by SIAKOTOS et al. (1969); 2’-3’ cyclic nucleotide-3’- study is very sensitive and 14% of the total activity TABLE1. PURITYOF Protein content or marker enzyme activities
Protein (mg/g wet wt) Lactate dehydrogenase (pmol NADH/min/g wet wt) Cytochrome oxidase (pg atom O/min/g wet wt) Acetylcholinesterase (pmol acetylcholine hydrolysed/ min/g wet wt) 2’-3’cyclic nucleotide 3‘-phosphohydrolase (pmol P,/min/g wet wt)
RAT BRAIN MYELIN FRACTIONS
Whole homogenate
Crude myelin fraction
+ 1.3
33.4 f 1.8 (9)
15.7 f 1-0 (10)
47.3 f 2.8 (4)
0 8 8 f 0.16 (4)
0
46.4 f 3.2 (5)
1.72 f 0.23 (4)
0.1 1 f 0.03 (5)
9.09 f 025(7)
1.24 & 0.04 (5)
0.06 f 0.01 (5)
+ 14
84.5 f 9.0 (6)
130.3
4 0 4 0 k 17
(23)
(5)
196.0
(5)
Purified myelin fraction
(4)
The figures in the table represent the protein content and enzyme activities found in the whole brain homogenate and myelin fractions derived from 1 g wet weight of brain. The enzyme and protein analysis was carried out as described in the text. Values are expressed as mean f S.E.M. with the number of observations in brackets.
Binding of triethyltin to myelin
o;I
1:
The equation for competitive binding of A at a single site in the presencc of a competing molecule B is v=
O.'
873
I I
2
3
4
+
nKArA1 KACAI
FIG.1. Binding of triethyltin to purified rat brain myelin. Thc solid line is that calculated for a 2 component system of binding sites 1 and 2 with thc following constants: n,, 0.54nmol/mg ofprotein; K I , 6.6 x lo5 M-'; n,, 13.5 nmol/ mg of protein; K,, 2.6 x 104M-'.
EDSALL& WYMAN (1958)
where V is the concentration of bound ligand/mg of protein, n is the concentration of total binding sites/ mg of protein, A is the concentration of free ligand and B is the concentration of free competing ligand. K A is the affinity constant for the binding of A and K, the affinity constant for the binding of B. With a range of concentrations of A and a fixed concentration of competitor B, where K' is the apparent affinity constant measured in the presence of B and the binding of A is measured V -
A
5
7(nrnol of triethyltin /rng of protein)
+ KBCB1
= K'(n
- vA).
Plotting VA/A against VA gives a line with a slope - K' and intercept of nK' on the y-axis and n on the x-axis (Fig. 2). The affinity constant K , may then be calculated from
K,
K, - K'
= ___
K"B1
When [B]/[A] is a constant (z), was found in the crude myelin but only 0.6% in the purified fraction. Having obtained a myelin fraction from rat brain which was free from major contaminating particles, the binding of triethyltin to this fraction was examined. Binding of trielhyltin to rut brain myelin A Scatchard analysis of the data for the binding of triethyl[113Sn]tin to purified myelin gave a curve (Fig. 1) indicating more than one class of binding sites (EDSALL& WYMAN, 1958; SCATCHARD, 1949). A satisfactory fit to the results may be obtained by a minimum of 2 classes of sites having constants of the value n , , 054 f 0.03 nmol/mg of protein; K , , 6.6 f 0.7 x 1 0 ' ~ ~ 'n2, ; 133 f 2.3 nmol/mg of protein; K,, 2.6 & 0.5 x lo4 M-' (mean of 7 experiments). The mean values of the constants obtained from 8 experiments using the crude myelin fraction were as follows: n,, 050 i 0.03 nmol/mg of protein; K , , 6.6 0.3 x lo5 M-'; nz, 14.1 f 1.1 nmol/mg of protein; K,, 2 3 f 0.1 x lo4 M-'. These results are identical with thosc found for the purified myelin and therefore for convenience the crude myelin fraction was used for subsequent experiments. Influence of other substances on the binding of triethyltin to rat brain myelin A number of other compounds were examined as competitors for the binding of triethyltin to the crude myelin fraction, and their binding constants determined from the results of these experiments.
V -
A
= nK, -
V,(KA
+ K, . z).
Then plotting P,JA against VA gives a line with slope - ( K A + K , .z) and an intercept of nKAon the y-axis and [n/(l K , .z/KA)] = n' on the x-axis (Fig. 2). From the experimentally determined values of the
+
nK
nK
Q, lh CCornpetitor I f ixed
n'
n
Y FIG.2. Diagrammatic representation of competitive binding at a single site (a) using a fixed concentration of competitor; where the number of binding sites is unaltered but the affinity constant is lowered; (b) using a constant ratio of competitor to triethyltin; where the number of binding sites is reduced and the affinity constant increases.
E. A. LOCK and W. N. ALDRIDGE
874
slope and total bound ligand A(n') the affinity constants K B for the competitor B may be calculated. Scatchard plots of experimental results of the binding of triethyl[113Sn]tin to rat brain myelin in the presence of 9.4 PM triethyl-lead or in the presence of a range of concentrations of triethyl-lead such that the ratio of triethyL1ead:triethyltin is 1.0 are shown in Figs. 3 and 4 respectively. They show examples of the general principles outlined above for the two experimental situations (cf Fig. 2) but for the more complicated situation where both triethyltin and triethyl-lead bind to two sites 1 and 2. When analysing a two-component system by curve fitting, the accuracy of the procedure is greatly influenced by the relative sues of n,, n2, K and K,. With the method using a constant concentration of competitor it becomes more difficult to separate a two-component system when the relative affinity constants are one order or less apart. For this reason the method using the constant ratio of competitor where the affinity constants increase is considered more accurate. The values for K , . measured in the presence of competitor, have been used to calculate KB. The binding of triethyl["3Sn]tin to rat brain myelin in the presence of hexachlorophane, 3,5-diiodo-4chlorosalicylanilide, trimethyltin and triethyl-lead was measured and the derived constants are tabulated in Table 2. Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilide had no observable effect on triethyltin binding to rat brain myelin. Trimethyltin competes with triethyltin for the high affinity binding site (1) in mye0.8
07
x
3.
=
\
c .m
c
05
2 R
r 0
-E
s
I I
I
I
2
3
I 4
I 5
B(nrnolof rriethyltin /rng ofprotein)
FIG.4. The effect of triethyl-lead on the binding of triethyltin to rat brain myelin. The solid lines are the best fit; analysis gave the following constants: Control (0) n,, 0,535nmol/mg of protein; KL.6 x lo5 M-'; n,, 10-4nmoli mg of protein; K , , 2 3 x 1 0 4 K 1 and triethyl-lead was present so that the [triethyl-lead]/[triethyltin] ratio was I : ] (0)n,, 042 nmol/mg of protein; K , . 9 x lo5 M - l ; n2. 6.67 nmol/mg of protein; K 2 , 3 x lo4 M -
lin. At a fixed concentration of trimethyltin the affinity for triethyltin is progressively lowered. whereas at a constant ratio of trimethyltin to triethyltin the affinity for triethyltin is increased (Table 2). An affinity constant of 2.1 f 0.5 x lo4 M - ' for trimethyltin binding to the high affinity site was calculated. Triethyl-lead also competes with triethyltin for the high affinity binding site (1) for myelin. At a fixed concentration of triethyl-lead the affinity for triethyltin is lowered, whereas at a constant ratio of triethyllead to triethyltin a progressive increase in affinity and decrease in number of triethyltin binding sites is observed (Table 2). The derived affinity constant for triethyl-lead was 2.7 f 0.5 x lo5 M - and is very similar to the affinity constant for triethyltin. DISCUSSION
0.4
k-l
a -
I
03
0.2
I
I
I
I
2
3
I 4
1 5
F ( n m o l of triethyltin /mg of protein) FIG. 3. The effect of triethyl-lead on the binding of triethyltin to rat brain myelin. The solid lines are the best fit; analysis gave the following constants: Control (0) n,, O-92nmol/mg of protein; K,, 5 x lo5 M - ' ; n,, 12.4nmoI/ mg of protein, K 2 , 2.5 x l O 4 K 1 and for triethyl-lead 9.4 pM (O), n,, 1.3 nmol/mg of protein, K,, 1 x lo5 M-'; n2. 20nmol/mg of protein; K , , 1 x 104hl-'.
Myelin was separated from rat brain and shown to be free from major contamination. Purified rat brain myelin binds triethyltin. The results when analysed by the method of SCATCHARD (1949) may be interpreted by a minimum of 2 classes of sites. One class of binding sites has an affinity of about 6.6 x lo5 M-' and a concentration of 0.54 nmol/mg of protein. The other class of site is of much lower affinity and higher concentration (2.6 x lo4 M - ' and 13.5 nmol/mg protein). Therefore triethyltin binds to rat brain myelin with an affinity similar to its affinity for rat haemoglobin (ROSE, 1969), for rat liver mitochondrial binding site (1) (ALDRIDGE& STREET,1970) and for a protein from guinea-pig liver supernatant fraction (ROSE & LOCK, 1970) (cf Table 3 for summary. The binding of triethyltin to rat haemoglobin and
Binding of triethyltin to myelin TABLE2. THEEFFECT OF
VARIOUS COMPOUNDS ON TH~.BINDINGOF TRIETHYLTIN TO RAT BRAIN MYELIN
"1
(nmol/mg of protein)
Compound Triethyltin (8) Trimethyltin
94pM 18-8pM
Trimeth Itin Triethitin
[
]
0.50 0.84 1.11
Triethyl-lead
K,
W1) 6.6
0.03
0.38
7'5
30
0 3 x lo5 5.0 105 3.0 105 _+
9.0
n2 (nmol/mg of protein)
(M-7
14.1 & 1.1 20.0 124
2.5 _+ 0.1 x lo4 2.0 x 104 2.5 x 104
105
9.8
10.0 x 10' 1.0 105
11.7 20.0
K2
KB
(M-
l)
2.1 x 104 3.5 x 104
3.5 x 104 3.0 x 104 1.0 104
1.3 x 104 4.2 x 105
1.3 x 104
94pM
0.55 1-30
1.0(2)
0.40
9.0 x lo5
3.0 x 105
0.33 0.08
18.0 x 105 80.0 x lo5
6.9 4.3 7.0
3.0 x lo4
1.5
3.0 x 104 1.0 104
1.6 x 105 1.5 x 105
9.7
3.0
50
[Hexachlorophane Triethyltin ] ''O 10.0
0.55
6.0 x 105
0.41
7.0
I ,o
0.44
[Salicylanilide] Triethyltin
875
104
105
3.0 x 104
For the preparation of myelin and the determination of binding see the Materials and Methods scction. The crude myelin fraction was used for these experiments. The results are single observations cxcept for triethyltin where they are expressed as mean f S.E.M.(No. of observations) n, and n2 are the concentration of binding sites I and 2; and K 1 and K , are their aRnity constants. K , is the derived affinity constant for the competitor at the high affinity binding site. It was calculated by knowing the K , for triethyltin and the K' for triethyltin in the presence of the competitor, see EDSALL& WYMAN(1958). guinea-pig liver supernatant fraction is sensitive to photo-oxidation, and the loss of binding was correlated with the loss of 2 histidine residues per binding site (ROSE, 1969; ROSE & LOCK, 1970). This finding is supported by the known chemistry of these compounds (POLLER, 1965; LUITJEN et al., 1962). In a single experiment, photo-oxidation of rat brain myelin fraction in the presence of methylene blue for 20min greatly reduced the number of high affinity triethyltin binding sites. On the basis of an analogy between the binding of triethyltin to other proteins, it is suggested that it binds to the protein moiety in myelin, and because of its sensitivity to photo-oxidation may 1961; WEILet involve histidine (RAY & KOSHLAND, a/.: 1951). The isolation and separation of 3 main types of protein from rat brain myelin has been fairIy well characterized (MARTENSON & GAITONDE, 1969; MEHL& HALARIS, 1970)and future study could establish whether triethyltin binds to 1 of these main protein classes. Triethyl-lead and trimethyltin compete with triTAULE 3. AFFINITY CONSTANTS FOR
Macromolecule Rat haemoglobin (ROSE,1968, 1969) Rat liver mitochondria (ALDRIDGE&
STREET,
THE BINDING OF TRIETHYLTIN IKIMETHYLTIN A N D TRIETHYL-LEAD TO MACROMOLECULES
Triethyltin
Affinity constant trimethyltin
3.5 x 1 0 5 ~ - 1
2.8
4.7 x 105 M - '
1.2
X
Triethyl-lead
1OsM-'
-
104~-1
-
-
-
1970)
Protein fraction from g. pig liver supernatant (ROSE& LOCK,1970) Rat brain myelin
2.0
X
106M-'
6.6 x 1 0 5 ~ - '
* Measured indirectly by competition with triethyltin. Not known.
ethyltin for the high affinity binding site in myelin. Triethyl-lead has a derived affinity constant for rat brain myelin of 2.7 x lo5 M - I (Table 3), very similar to that of triethyltin whereas trimethyltin has a thirtyfold lower affinity constant than triethyltin at 2.1 x lo4 M-' (Table 3). The affinity constant for the binding of trimethyltin to rat liver mitochondria is also about thirty times lower than that for triethyltin (ALDRIDCE & STREET,1970) (Table 3). Neither triethyl-lead nor trimethyltin produces an oedematous lesion in the central nervous system (BARNES& STONER, 1958; CREMER, 1959). Hexachlorophane and 3,5-diiodo-4-chlorosalicylanilided o not compete with tricthyltin for the high affinity binding site in myelin. However, both these compounds produce an oedematous lesion in the central nervous system similar to triethyltin (KIMBROUGH & GAINES,1971 ; KURTZet al., 1969; LOCK, 1974). Therefore the high affinity binding to rat brain myelin either is not involved or is not the only factor involved in the production of oedema by these compounds.
2.1
x
1 o 4 ~ - I *
2.7 x 105 M-
'*
876
E. A. LOCKand W. N. ALDRIDGE
LQCKE. A. (1974) Ph.D. 7hesis, CNAA. LUUTENJ. G. A.: JANSSENM. J. & VAN DER KERKG. J. M. (1962) Reel Trav. chim. Pay-Bas Belg. 81. 202-205. REFERENCES MACFEP. N., STONERH. B. & BARNESJ. M. (1957) J. path. Bact. 73. 107-124. B. W. (1970) Biochem. J . 118. ALDRIDGE W. N. & STREET MEHLE. & HALARJS A. (1970) J. Neurochem. 17. 659-668. 171-1 79. R. E. & GAITONDE M. K. (1969) J. Neurochem. ALDRIDGEW. N., EMERYR. C. & STREETB. W. (1960) MARTENSON 16. 889-898. Biochem. J. 77, 326-327. ALEUF. P., KATZMAN R. & TERRY R. D. (1963) J . Neuro- NORTONW. T . (1971) Adv. exp. med. B i d . 13. 327--337. OLAFSON R. W., DRUMMOND G. 1. & LEE J. F. (1969) Can. path. exp. Neurol. 22. 403413. J . Biochem. 47. 961-966. BARNESJ. M. & STONER H. B. (1958) Br. J. industr. Med. 15. 1 s 2 2 . POLLER R. C. (1965) J . Organornet. Chear. 3. 321-329. RAYW. J. & KOSHLANDD. E. (1961) J . bid. Chem. 236. BERENBLUM I. & CHAINE. (1938) Biochem. J . 32. 286-298. BERGMEYER 1973-1 979. H. (1963) Methods of Enzymatic Analysis. Academic Press, New York. ROSE M. S . (1968) Ph.D. Thesis, University of London. CREMER J . E. (1959) Br. J . industr. Med. 16. 191-199. ROSEM. S. (1969) Biochem J . 111. 129--137. EDSALLJ. T . & WYMANJ. (1958) Biophysical Chemistry, ROSEM. S . & ALDRIDCEW. N. (1968) Biochem. J . 106. Vol. 1, pp. 651-653. Academic Press, New York. 821-828. ETO Y., SUZUKIK. & SuzuK1 K. (1971) J. Lipid Res. 12. ROSEM. S . & LOCKE. A. (1970) Biochem. J. 120. 151-157. 570-579. SCATCHARD G. (1949) Ann. N.Y. Acad. Sci. 51. 660-672 GORNALL A. K., BARDAWILL C. J. & DAVIDM. M. (1949) SCHNAITMAN J. W. (1967) C., ERWINV. G. & GREENAWALT J . bid. Chem. 177. 751-766. J . Cell. B i d . 32. 719-735. HESSH. H. & LEWINE. (1965) J. Neurochem. 12. 205-21 1. SMKOTOSA. N., FILBERT M. & HESTER R. (1969) Biochem. KIMBROUGH R. D. & GAIN&T . B. (1971) Archs enuiron. Med. 3. 1-12. Health 23. 114-118. SMITH M. E. (1973) J. Neurochem. 21. 357-372. KURIHARAT. & TSUKADA Y. (1967) J. Neurochrm. 14. THRELFALL C. J. (1957) Bwchem. J . 65. 694699. 1167-1 174. WEBSTER G. R. & SMITHA. T. (1964) Biochem. J. 90, 64-65. KURTZS.M., SCHARDEIN J. L., FITZGERALD J. E. & KAUMP WEIL L., GORDON W. G. & BUCHERTA. R. (1951) Archs D. H. (1969) Tox. appl. Pharmac. 14. 652. Biochem. Biophys. 33. 90-109. LEE J. C. & BAKAYL. (1965) Archs Neurol. Psychiat. Chicago 13, 48-57. Acknowledgement-The authors thank Dr W. H. BUTLERfor the electron microscopy on the myelin fractions.
