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In vitro synthesis of sulfatide in a myelin deficient mutant: The Jimpy mouse
BRAINRESEARCH
433
I N VITRO SYNTHESIS OF SULFATIDE IN A MYELIN DEFICIENT MUTANT:
THE JIMPY MOUSE
J.-M. MATTHIEU, U. SCHNEIDER AND N. HERSCHKOWITZ Department of Pediatrics, University of Berne, Berne (Switzerland)
(Accepted January 19th, 1972)
INTRODUCTION The Jimpy mutation in the mouse results in an inheritable defect of myelin synthesis which leads to greatly decreased myelination in the central nervous systemS,9,10,~1,~. Biochemical investigations have shown both a decreased cerebroside and a decreased sulfatide content in the brain6,7,9,11,19,21 caused by a deficiency of several enzymes which are mainly localized in myelin 16-1s. Other enzyme defects involving myelination have been reported4,12-14. Cerebroside sulfotransferase (CST), the enzyme which transfers sulfate from adenosine 3'-phosphate-5'-phosphosulfate (PAPS) to cerebroside to form sulfatide 1-3, 15, shows decreased activity in the central nervous system of the Jimpy mouse, but is normal in the peripheral nervous system 9. The pH optimum, heat lability 20 and Michaelis constant of an enzyme are dependent on its protein structure. Using these parameters the residual activity of CST from Jimpy brain has been compared with that of normal brain, peripheral nervous system and kidney. Abbreviations and terminology: ATP, adenosine-5-triphosphate; Brij-96®, polyoxyethylene-10 oleyl ether; Cerebroside, galactosylceramide; CST, cerebroside sulfotransferase; PAPS, adenosine Y-phosphate-5'-phosphosullate; Psychosine, galactosylsphingosine; Sulfatide, sulfogalactosylceramide. MATERIALSAND METHODS Materials
Adenosine-5-triphosphate (ATP) was obtained from Boehringer GmbH, Mannheim, Germany. One per cent Brij-96® (polyoxyethylene-10 oleyl ether) was obtained from Atlas Chemical Industries, Wilmington, Del., U.S.A. Cerebroside (bovine brain grade II) was obtained from Sigma Chemical Company, St. Louis, Mo., U.S.A. [asS]Na2SO4, 1 mCi/ml was obtained from Eidg. Institut ffir Reaktorforschung, Isotopenproduktion, Wiirenlingen, Switzerland. [asS]PAPS was prepared according to McBrain Research, 42 (1972) 433-439
434
J.-M. MATTHIEUet al.
Khann and Ho 15. Other materials were of analytical grade and purchased from E. Merck, Darmstadt, Germany. Animals Male mutants from the Jimpy strain, C57 Bc/6J-AW-J-Ta+/+jp, which originated at the Jackson Laboratories, Bar Harbor, Maine, U.S.A., were used as experimental animals. The control animals were of the same strain but not carrying the mutant gene. Tests and controls were matched by age, body weight and sex. They were fed with Miuse-Ratten-Zuchtwiirfel No 184, NAFA, Gossau, Switzerland, and tap water ad libitum. Enzyme preparation A 20% homogenate in 0.25 M sucrose from 17-day-old mouse brains was prepared in an all glass homogenizer. The homogenate was centrifuged at t000 x g for I0 min at 4 °C, and the resulting supernatant was used as the source of crude enzyme15. Enzymatic assay The incubation mixture consisted of 199.7 #M imidazole buffer, with the pH adjusted to the appropriate values with HC1; 16 btM ATP; 9.9 # M MgCI2; 1.6 # M KC1; 1.09 # M cerebroside in 1% Brij-96; 600,000 disint./min of [asS]PAPS; 0.1 ml of crude enzyme (-4- 2 mg protein); and distilled water to a final volume of 1.2 ml. Duplicate samples were incubated (unless otherwise stated) for 120 min at 37 °C. The pH of the reaction mixture was checked at the beginning and at the end of 120 min of incubation. The reaction was stopped by addition of 12 ml chloroformmethanol (1:1, v/v). [35S]Sulfatide was extracted as previously describeda. The lipid extracts were taken to dryness under a stream of N2 in liquid scintillation counting vials. To each vial was added 10 ml of a toluene scintillation counting mixture: Pre mix 'P' from Packard (2,5-diphenyloxazole (PPO), 98% and 1,4-bis-2-(5-phenyloxazolyl)benzene (POPOP), 2 %) 4.0 g/liter of toluene. The radioactivity of samples was counted in a Packard Tri-Carb liquid scintillation spectrometer, model 3380. The values, corrected for quenching and counting efficiency, are expressed in disint./min. RESULTS Unless otherwise stated, the results are expressed in pca-centages of the maximum value obtained in the same experiment. Each different condition (pH o r incubation time at 45 °C) was investi~tted in duplicate, the e x p e ~ n t boing rgl~tted a t least twice. Brain Research, 42 (1972) 433-439
435
SULFATIDE SYNTHESIS IN JIMPY MICE
~- 5.0g ,~, o-~
E ¢-
"~
normal Jimpy
t,.O_
/
/
./ / /
/ /
Ot
E "Tw
/
3.0-
/
4
/ /
•
~'¢
2.0_
~
1.0
o/O'~ /
1/S
( m M "1 )
Fig. 1. Effect of cerebroside concentration on cerebroside sulfotransferase activity.
-
--0
E
normal
c
1.0-
"
0.5_
~-~
J i m py
, , ,.";'
s "e I s I ' ' e ' S
I
I
I
I
2 4 e 8 1'o
1'4 16
1/S (.uc35S-PAPS-1 m l ) Fig. 2. Effect of [asS]PAPS concentration on cerebroside sulfotransferase activity.
Cerebroside sulfotransferase activity in brain Using Jimpy or normal mouse brain, the amount of [85S]sulfatide synthesized was proportional to the concentration of exogenous cerebroside (Fig. 1) or [85S]PAPS (Fig. 2). The specific enzyme activity was much lower in Jimpy brain than in control mice; both curves had the same origin (Figs. 1 and 2). The Lineweaver-Burk plot was used to calculate the apparent Km of 4.5 • 10-4 M for cerebroside which was the same in control and Jimpy brain. The pH optimum of CST was found to be 7.0 (Fig. 3) for both control and Jimpy brain. Exposure of the incubation mixture at 45 °C for increasing lengths of time Brain Research, 42 (1972) 433-439
436
J.-M. MATTHIEUe t a / .
*/,ACTIVITY BRAIN 100 ,.~,. .-, r~ normal (N=4) . . . . . py - ) 8 0 ] ~ ~ ' ~t , ~ ; - e m Jim (N-3
60J
,
*\
0
6.0
7:0
8~
910 pH
Fig. 3. Effect of pH on cerebroside sulfotransferase in Jimpy and normal brain, Each point is the mean of 3 and 4 independent experiments respectively. Data are given as percentages of the maximum observed value (pH optimum).
lOO~
%ACTIVITY
801 ~ i
BRAIN --o r~(N=3) normal
w
60~~ ~~
--* . . . .
4oi ~ 20.
Jimpy
~5"C
~,~
o
0 3 5 7 10
20
30.Min.
Fig. 4. Effect of heat inactivation (45 °C) on cerebrosid¢su]fotransfera~¢activity. showed the same inactivation kinetics for both control and Jimpy brain (Fig. 4). Seven min exposure decreased the enzymatic activity to 24% (Table I); by 30 min practically no activity could be detected. Cerebroside sulfotransferase activity in sciatic nerve
There was no difference in the a m o u n t o f [asS]sulfatide synthesized by control Brain Research, 42 (1972) 433-439
SULFATIDESYNTHESISIN JIMPY MICE
437
TABLE I CEREBROSIDE SULFOTRANSFERASE
pH optimum and residual activity (after a 7 min exposure to 45 °C) of cerebrosidesulfotransferase from control animals. Means of 3-5 independentexperiments(4- S.E.M.). Organ
pH optimum Heat inactivation (7 min at 45 °C)
Brain
Sciatic nerve
7.0
6.8
24.0% 4-5 %
5.5 % -4-0.5%
Kidney
8.0 6.9 44.0% 4-3 %
and Jimpy sciatic nerves. The pH optimum and heat lability of the enzyme activity in sciatic nerve, however, were different from that of brain. The residual activity of CST after 7 min exposure at 45 °C was 5.5%. No activity could be detected after a preincubation period of 10 min at 45 °C (Table I). Cerebroside sulfotransferase activity in kidney
Jimpy kidney preparation showed a normal rate of in vitro [35S]sulfatide synthesis. Characterization of CST in control kidney, however, revealed quite different properties when compared to that of brain or sciatic nerve: the pH curve showed two different optima at 6.9 and 8.0; after a 7 min exposure to 45 °C, the residual enzyme activity was still 44.0% (Table I). DISCUSSION The residual activity of CST in the mutant Jimpy brain showed a normal pH optimum, heat stability and Km value for cerebroside and PAPS when compared to normal brain. There was therefore no evidence that the protein structure of the enzyme was altered in the Jimpy mutant. The low enzyme activity may be caused either by decreased synthesis or by increased degradation of a normal enzyme protein, inhibition of the enzyme activity having been ruled out by previous experiments9. These results are in agreement with those of Neskovic et al.17 for galactosylsphingosine transferase in normal and Jimpy brain. Jimpy sciatic nerve showed normal enzyme activity in comparison with the enzyme activity in non-mutant mice, and in contrast to the lowered activity in Jimpy brain. Comparing CST characteristics in brain, sciatic nerve and kidney, 3 different pH optima and 3 different labilities to heat inactivation (Table I) were found. These results are consistent with the postulate that the protein structures of CST in brain and the other organs differ from each other and that they are under different genetic control. In the Jimpy mouse, CST in brain only is affected by the mutation. The differences of CST in brain and sciatic nerve are a further indication that myelinBrain Research, 42 (1972) 433-439
438
J.-M. MATTHIEU el ~tl.
a t i o n in the central nervous system is u n d e r specific genetic c o n t r o l separate from t h a t g o v e r n i n g myelination o f the p e r i p h e r a l n e r v o u s system. SUMMARY (1) T h e / n vitro synthesis o f [a~S]sulfatide f r o m [35S]PAPS, using exogenous c e r e b r o s i d e as the sulfate a c c e p t o r , was studied in J i m p y a n d c o n t r o l mice. (2) J i m p y b r a i n exhibited a low c e r e b r o s i d e sulfotransferase activity. E n z y m i c kinetic studies showed no differences for Km, p H o p t i m u m a n d heat inactivation in c o n t r o l a n d J i m p y brain. (3) Sulfatide synthesis in J i m p y p e r i p h e r a l n e r v o u s system a n d kidney was f o u n d to be n o r m a l , p H o p t i m u m a n d heat i n a c t i v a t i o n o f C S T differed from each o t h e r in brain, p e r i p h e r a l n e r v o u s system a n d kidney, which was consistent with the p r o p o sition t h a t the e n z y m e p r o t e i n s in these o r g a n s differ f r o m each o t h e r a n d t h a t they are u n d e r different genetic control. O n l y the e n z y m e l o c a t e d in the b r a i n seemed to be affected by the J i m p y m u t a t i o n . ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n , G r a n t No. 3.145.69.
REFERENCES 1 BALASUBRAMANIAN,A. S., AND BACHHAWAT, B. K., Formation of cerebroside sulfate from 3'-
2
3 4 5 6 7 8 '9 10 11
phosphoadenosine 5'-phosphosulfate in sheep brain, Biochim. biophys. Acta (Amst.), 106 (1965) 218-220. BALASUBRAMANIAN,A. S., AND BACHHAWAT, B. K., Studies on enzymic synthesis of cerebroside sulfate from 3'-phosphoadenosine 5'-phosphosulfate, Ind. J. Biochem., 2 (1965) 212-216. CHASE,H. P., DORSEY,J., AND McKnANN, G. M., The effect of malnutrition on the synthesis of a myelin lipid, Pediatrics, 40 (1967) 551-559. DF.SHMUKH,D. S., INOUE,T., AND PIERINGER, R. A., The association of the galactosyl diglycerides of brain with myelination. II. The inability of the myelin-deficient mutant, Jimpy mouse, to synthesize galactosyl diglycerides effectively, J. biol. Chem., 246 (1971) 5695-5699. FARKAS,E., ZAHND, J. P., NUSSBAUM,J. L., ET MANDEL, P., Etude histologique, histochimique et ultrastructurale des souris de souche Jimpy, Coil. Int. CNRS, 924 (1970)21-27. GALLI,C., KNEFaONE,G. M., AND PAOLETrt, R., An inborn error of cerebroside biosynthesis as the molecular defect of the Jimpy mouse brain, Life Sci., 8 (1969) 911-918. GALLI, C., AND RE CECCONI GALU, D., Cerehroside and sutphatide deficiency in the brain of 'Jimpy mice', a mutant strain of mice exhibiting neurological symptoms, Nature (Lond.), 220 (1968) 165-166. HERSCHKowrrz, N., MCKHANN, G. M., SAXENA,S., AND SHOOTER, E. M., Characterization of sulphatide-containing iipoproteins in rat brain, J. Neuroehem., 15 (1968) 1181-1188. HERSCHKOWrrz,N., VA~ELLA,F., ANDBmCHOFF,A., Myelin differences in the central and periph. eral nervous system in the 'Jimpy mouse', J. Neurochem., 18 (1971) 1361-1363. HIRANO,A., SAX,D. S., ANDZIMMBRMAN,H. M., The fine structure of the cerebella of Jimpy mice and their 'normal' litter mates, J. Neuropath. exp. Neurol., 28 (1969) 388-400. HOGAN,E. L., JOSEPH, K. C., ANDSCHMIDT,G., Composition of cerebral lipids in murine sudanophilic leucodystrophy: the Jimpy mutant, J. Neurochem., 17 (1970) 75-83.
Brain Research, 42 (1972) 433-439
SULFATIDE SYNTHESIS IN JIMPY MICE
439
12 KURII-IARA,T., NUSSBAUM,J. L., AND MANDEL,P., 2',3'-Cyclic nucleotide 3'-phosphohydrolase in the brain of the 'Jimpy' mouse, a mutant with deficient myelination, Brain Research, 13 (1969) 401-403. 13 KURIHARA,T., NUSSBAUM,J. L., AND MANDEL,P., 2',3'-Cyclic nucleotide 3'-phosphohydrolase in brains of mutant mice with deficient myelination, J. Neurochem., 17 (1970) 993-997. 14 KURIHARA,T., NUSSaAUM,J. L., AND MANDEL,P., 2',3'-Cyclic nucleotide 3'-phosphohydrolase in purified myelin from brain of Jimpy and normal young mice, Life Sci., 10 (1971) 421-429. 15 McKnANN, G. M., AND HO, W., The in vivo and in vitro synthesis of sulphatides during development, J. Neurochem., 14 (1967) 717-724. 16 NF~SKOV/¢,N. M., NUSSBAUM,J. L., AND MANDEL, P., Enzymatic synthesis of psychosine in 'Jimpy' mice brain, FEBS Letters, 3 (1969) 199-201. 17 NESKOVIC,N., NUSSBAUM,J. L., AND MANDEL,P., Enzymatic deficiency in neurological mutants. Brain uridine diphosphate galactose:ceramide galactosyl transferase in Jimpy mouse, FEBS Letters, 8 (1970) 213-216. 18 NESKOVIC,N. M., NUSSaAUM,J. L., AND MANDEL,P., A study of glycolipid metabolism in myelination disorder of Jimpy and Quaking mice, Brain Research, 21 (1970) 39-53. 19 NUSSaAUM,J. L., NESKOVIC,N., AND MANDEL,P., A study of lipid components in brain of the 'Jimpy' mouse, a mutant with myelin deficiency, J. Neurochem., 16 (1969) 927-934. 20 PAIGEN,K., The genetics of enzyme realization. In M. RECHCIGL,JR. (Ed.), Enzyme Synthesis and Degradation in Mammalian Systems, Karger, Basel, 1971, p. 8. 21 SIDMAN, R. L., DICKIE, M. M., AND APPEL, S. H., Mutant mice (Quaking and Jimpy) with deficient myelination in the central nervous system, Science, 144 (1964) 309-311. 22 TORII,J., ADACHI,M., AND VOLK, B. W., Histochemical and ultrastructural studies of inherited leukodystrophy in mice, J. Neuropath. exp. Neurol., 30 (1971) 278-289.
Brain Research, 42 (1972) 433-439
433
I N VITRO SYNTHESIS OF SULFATIDE IN A MYELIN DEFICIENT MUTANT:
THE JIMPY MOUSE
J.-M. MATTHIEU, U. SCHNEIDER AND N. HERSCHKOWITZ Department of Pediatrics, University of Berne, Berne (Switzerland)
(Accepted January 19th, 1972)
INTRODUCTION The Jimpy mutation in the mouse results in an inheritable defect of myelin synthesis which leads to greatly decreased myelination in the central nervous systemS,9,10,~1,~. Biochemical investigations have shown both a decreased cerebroside and a decreased sulfatide content in the brain6,7,9,11,19,21 caused by a deficiency of several enzymes which are mainly localized in myelin 16-1s. Other enzyme defects involving myelination have been reported4,12-14. Cerebroside sulfotransferase (CST), the enzyme which transfers sulfate from adenosine 3'-phosphate-5'-phosphosulfate (PAPS) to cerebroside to form sulfatide 1-3, 15, shows decreased activity in the central nervous system of the Jimpy mouse, but is normal in the peripheral nervous system 9. The pH optimum, heat lability 20 and Michaelis constant of an enzyme are dependent on its protein structure. Using these parameters the residual activity of CST from Jimpy brain has been compared with that of normal brain, peripheral nervous system and kidney. Abbreviations and terminology: ATP, adenosine-5-triphosphate; Brij-96®, polyoxyethylene-10 oleyl ether; Cerebroside, galactosylceramide; CST, cerebroside sulfotransferase; PAPS, adenosine Y-phosphate-5'-phosphosullate; Psychosine, galactosylsphingosine; Sulfatide, sulfogalactosylceramide. MATERIALSAND METHODS Materials
Adenosine-5-triphosphate (ATP) was obtained from Boehringer GmbH, Mannheim, Germany. One per cent Brij-96® (polyoxyethylene-10 oleyl ether) was obtained from Atlas Chemical Industries, Wilmington, Del., U.S.A. Cerebroside (bovine brain grade II) was obtained from Sigma Chemical Company, St. Louis, Mo., U.S.A. [asS]Na2SO4, 1 mCi/ml was obtained from Eidg. Institut ffir Reaktorforschung, Isotopenproduktion, Wiirenlingen, Switzerland. [asS]PAPS was prepared according to McBrain Research, 42 (1972) 433-439
434
J.-M. MATTHIEUet al.
Khann and Ho 15. Other materials were of analytical grade and purchased from E. Merck, Darmstadt, Germany. Animals Male mutants from the Jimpy strain, C57 Bc/6J-AW-J-Ta+/+jp, which originated at the Jackson Laboratories, Bar Harbor, Maine, U.S.A., were used as experimental animals. The control animals were of the same strain but not carrying the mutant gene. Tests and controls were matched by age, body weight and sex. They were fed with Miuse-Ratten-Zuchtwiirfel No 184, NAFA, Gossau, Switzerland, and tap water ad libitum. Enzyme preparation A 20% homogenate in 0.25 M sucrose from 17-day-old mouse brains was prepared in an all glass homogenizer. The homogenate was centrifuged at t000 x g for I0 min at 4 °C, and the resulting supernatant was used as the source of crude enzyme15. Enzymatic assay The incubation mixture consisted of 199.7 #M imidazole buffer, with the pH adjusted to the appropriate values with HC1; 16 btM ATP; 9.9 # M MgCI2; 1.6 # M KC1; 1.09 # M cerebroside in 1% Brij-96; 600,000 disint./min of [asS]PAPS; 0.1 ml of crude enzyme (-4- 2 mg protein); and distilled water to a final volume of 1.2 ml. Duplicate samples were incubated (unless otherwise stated) for 120 min at 37 °C. The pH of the reaction mixture was checked at the beginning and at the end of 120 min of incubation. The reaction was stopped by addition of 12 ml chloroformmethanol (1:1, v/v). [35S]Sulfatide was extracted as previously describeda. The lipid extracts were taken to dryness under a stream of N2 in liquid scintillation counting vials. To each vial was added 10 ml of a toluene scintillation counting mixture: Pre mix 'P' from Packard (2,5-diphenyloxazole (PPO), 98% and 1,4-bis-2-(5-phenyloxazolyl)benzene (POPOP), 2 %) 4.0 g/liter of toluene. The radioactivity of samples was counted in a Packard Tri-Carb liquid scintillation spectrometer, model 3380. The values, corrected for quenching and counting efficiency, are expressed in disint./min. RESULTS Unless otherwise stated, the results are expressed in pca-centages of the maximum value obtained in the same experiment. Each different condition (pH o r incubation time at 45 °C) was investi~tted in duplicate, the e x p e ~ n t boing rgl~tted a t least twice. Brain Research, 42 (1972) 433-439
435
SULFATIDE SYNTHESIS IN JIMPY MICE
~- 5.0g ,~, o-~
E ¢-
"~
normal Jimpy
t,.O_
/
/
./ / /
/ /
Ot
E "Tw
/
3.0-
/
4
/ /
•
~'¢
2.0_
~
1.0
o/O'~ /
1/S
( m M "1 )
Fig. 1. Effect of cerebroside concentration on cerebroside sulfotransferase activity.
-
--0
E
normal
c
1.0-
"
0.5_
~-~
J i m py
, , ,.";'
s "e I s I ' ' e ' S
I
I
I
I
2 4 e 8 1'o
1'4 16
1/S (.uc35S-PAPS-1 m l ) Fig. 2. Effect of [asS]PAPS concentration on cerebroside sulfotransferase activity.
Cerebroside sulfotransferase activity in brain Using Jimpy or normal mouse brain, the amount of [85S]sulfatide synthesized was proportional to the concentration of exogenous cerebroside (Fig. 1) or [85S]PAPS (Fig. 2). The specific enzyme activity was much lower in Jimpy brain than in control mice; both curves had the same origin (Figs. 1 and 2). The Lineweaver-Burk plot was used to calculate the apparent Km of 4.5 • 10-4 M for cerebroside which was the same in control and Jimpy brain. The pH optimum of CST was found to be 7.0 (Fig. 3) for both control and Jimpy brain. Exposure of the incubation mixture at 45 °C for increasing lengths of time Brain Research, 42 (1972) 433-439
436
J.-M. MATTHIEUe t a / .
*/,ACTIVITY BRAIN 100 ,.~,. .-, r~ normal (N=4) . . . . . py - ) 8 0 ] ~ ~ ' ~t , ~ ; - e m Jim (N-3
60J
,
*\
0
6.0
7:0
8~
910 pH
Fig. 3. Effect of pH on cerebroside sulfotransferase in Jimpy and normal brain, Each point is the mean of 3 and 4 independent experiments respectively. Data are given as percentages of the maximum observed value (pH optimum).
lOO~
%ACTIVITY
801 ~ i
BRAIN --o r~(N=3) normal
w
60~~ ~~
--* . . . .
4oi ~ 20.
Jimpy
~5"C
~,~
o
0 3 5 7 10
20
30.Min.
Fig. 4. Effect of heat inactivation (45 °C) on cerebrosid¢su]fotransfera~¢activity. showed the same inactivation kinetics for both control and Jimpy brain (Fig. 4). Seven min exposure decreased the enzymatic activity to 24% (Table I); by 30 min practically no activity could be detected. Cerebroside sulfotransferase activity in sciatic nerve
There was no difference in the a m o u n t o f [asS]sulfatide synthesized by control Brain Research, 42 (1972) 433-439
SULFATIDESYNTHESISIN JIMPY MICE
437
TABLE I CEREBROSIDE SULFOTRANSFERASE
pH optimum and residual activity (after a 7 min exposure to 45 °C) of cerebrosidesulfotransferase from control animals. Means of 3-5 independentexperiments(4- S.E.M.). Organ
pH optimum Heat inactivation (7 min at 45 °C)
Brain
Sciatic nerve
7.0
6.8
24.0% 4-5 %
5.5 % -4-0.5%
Kidney
8.0 6.9 44.0% 4-3 %
and Jimpy sciatic nerves. The pH optimum and heat lability of the enzyme activity in sciatic nerve, however, were different from that of brain. The residual activity of CST after 7 min exposure at 45 °C was 5.5%. No activity could be detected after a preincubation period of 10 min at 45 °C (Table I). Cerebroside sulfotransferase activity in kidney
Jimpy kidney preparation showed a normal rate of in vitro [35S]sulfatide synthesis. Characterization of CST in control kidney, however, revealed quite different properties when compared to that of brain or sciatic nerve: the pH curve showed two different optima at 6.9 and 8.0; after a 7 min exposure to 45 °C, the residual enzyme activity was still 44.0% (Table I). DISCUSSION The residual activity of CST in the mutant Jimpy brain showed a normal pH optimum, heat stability and Km value for cerebroside and PAPS when compared to normal brain. There was therefore no evidence that the protein structure of the enzyme was altered in the Jimpy mutant. The low enzyme activity may be caused either by decreased synthesis or by increased degradation of a normal enzyme protein, inhibition of the enzyme activity having been ruled out by previous experiments9. These results are in agreement with those of Neskovic et al.17 for galactosylsphingosine transferase in normal and Jimpy brain. Jimpy sciatic nerve showed normal enzyme activity in comparison with the enzyme activity in non-mutant mice, and in contrast to the lowered activity in Jimpy brain. Comparing CST characteristics in brain, sciatic nerve and kidney, 3 different pH optima and 3 different labilities to heat inactivation (Table I) were found. These results are consistent with the postulate that the protein structures of CST in brain and the other organs differ from each other and that they are under different genetic control. In the Jimpy mouse, CST in brain only is affected by the mutation. The differences of CST in brain and sciatic nerve are a further indication that myelinBrain Research, 42 (1972) 433-439
438
J.-M. MATTHIEU el ~tl.
a t i o n in the central nervous system is u n d e r specific genetic c o n t r o l separate from t h a t g o v e r n i n g myelination o f the p e r i p h e r a l n e r v o u s system. SUMMARY (1) T h e / n vitro synthesis o f [a~S]sulfatide f r o m [35S]PAPS, using exogenous c e r e b r o s i d e as the sulfate a c c e p t o r , was studied in J i m p y a n d c o n t r o l mice. (2) J i m p y b r a i n exhibited a low c e r e b r o s i d e sulfotransferase activity. E n z y m i c kinetic studies showed no differences for Km, p H o p t i m u m a n d heat inactivation in c o n t r o l a n d J i m p y brain. (3) Sulfatide synthesis in J i m p y p e r i p h e r a l n e r v o u s system a n d kidney was f o u n d to be n o r m a l , p H o p t i m u m a n d heat i n a c t i v a t i o n o f C S T differed from each o t h e r in brain, p e r i p h e r a l n e r v o u s system a n d kidney, which was consistent with the p r o p o sition t h a t the e n z y m e p r o t e i n s in these o r g a n s differ f r o m each o t h e r a n d t h a t they are u n d e r different genetic control. O n l y the e n z y m e l o c a t e d in the b r a i n seemed to be affected by the J i m p y m u t a t i o n . ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by the Swiss N a t i o n a l F o u n d a t i o n , G r a n t No. 3.145.69.
REFERENCES 1 BALASUBRAMANIAN,A. S., AND BACHHAWAT, B. K., Formation of cerebroside sulfate from 3'-
2
3 4 5 6 7 8 '9 10 11
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