- Email: [email protected]
Different responses of the hepatic and erythropoietic σ-aminolevulinic acid synthetase of mice
SHORT COMMUNICATIONS
585
will probably be most useful in studying conformational changes in synthetic polypeptides where only one reactive functional group is present. We are pleased to thank Professors S. A. BERNHARD and A. R. LARRABEE for helpful discussions and to National Science Foundation (Grant GP-55o9) for support of this work. D.L.N. was a National Science Foundation undergraduate research participant, summer 1965 .
Department of Chemistry, University of Oregon, Eugene, Oreg. 97403 (U.S.A.)
O. H . G R I F F I T H J. F. W. KEANA
D. L. NOALL J. L. IVEY
I T. J. STONE,T. BUCKMAN,13. L. NORDIO AND H. M. MCCONNELL, Proc. Natl. Acad. Sci. U.S., 54 (1965) iOlO. 2 L. STRYER ANn O. H. GRIFFITH, Proc. Natl. Acad. Sci. U.S., 54 (1965) 1785. 3 A. S. WAGGONER,O. H. GRIFFITH AND C. R. CHRISTENSEN, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1198. 4 S. OGAWA AND H. M. MCCONNELL, Proc. Natl. Acad. Sci. U.S., 58 (1967) 19. 5 E. G. ROZANTZI~V AND L. A. KRINITZKAYA, Tetrahedron, 21 (1965) 491. 6 D. S. TARBELL AND N. A. LglSTER, J. Org. Chem., 23 (1958) 1149. 7 T. B. WINDHOLZ, J. Org. Chem., 23 (1958) 2044. 8 T. B. WINDI~OLZ, J. Org. Chem., 25 (196o) 17o 3. 9 D. S. TARBlgLL AND E. J. LONGOSZ, J. Org. Chem., 24 (1959) 774. i o A. G. GORNALL, C. J. BARDAWILL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. i i N. A. LEISTER AND D. S. TARBELL, J. Org. Chem., 23 (1958) 1152.
Received August I8th, i967 Biochim. Biophys. Acta, 148 (1967) 583-585
BBA
2337o
Different responses of the hepatic and erythropoietic ~i-aminolevulinic acid
synthetase of mice ~-Aminolevulinie acid synthetase demonstrated in some species of microorganisms1, 2 and also in the livez a and erythropoietic organs 4 of mammals may be regarded as the rate-limiting enzyme in porphyrin and heine biosynthesis. Furthermore, a number of indirect observations 5 has suggested that the state of cellular oxidation might influence the metabolic regulation of porphyrin and heme biosynthesis through the activity of S-aminolevulinic acid synthetase. In microorganisms in fact, ~-aminolevulinic acid synthetase activity is shown to be regulated by oxygen and heme concentrations in the environment1,2,6. Furthermore, it has been shown by GRANICK~ with chick embryo liver cells and more recently by TSCHUDYs in rats, that hemin acts as a corepressor to prevent the formation of ~-aminolevulinic acid synthetase in the liver. The present experiments were designed to study the responses of the 3-aminolevulinic acid synthetase activity in the liver and erythropoietic organ of male mice (D-D strain, weighing 18-2o g) to hypoxia, polycythemia, erythropoietin and 3,5-dicarbethoxy-i,4-dihydroeollidine (DDC) as a porphyrinogenic substance. As the erythropoietic organ, we chose the spleen which has been shown to be analogous A b b r e v i a t i o n : DDC, 3 , 5 - d i c a r b e t h o x y - I , 4 - d i h y d r o c o l l i d i n e .
Biochim. Biophys. Acta, 148 (I967) 585-587
5~6
SHORT COMMUNICATIONS
t o b o n e m a r r o w f r o m t h e s t a n d p o i n t of e r y t h r o p o i e s i s i n m i c e 9. L i v e r m i t o c h o n d r i a w e r e p r e p a r e d a c c o r d i n g t o t h e m e t h o d of HOGEBOOM AaD SCHNEIDER ~°. F o r t h e p r e p a r a t i o n of m i t o c h o n d r i a f r o m s p l e n i c cells, t h e s p l e e n s o b t a i n e d f r o m 5 m i c e w e r e c r u s h e d a n d s u s p e n d e d i n 5 m l of c o o l e d i s o l o g o u s p l a s m a a n d p a s s e d t h r o u g h a fine m e s h . C o u n t s of n u c l e a t e d cells a n d r e t i c u l o c y t e s , a n d p r e p a r a t i o n of s m e a r s f r o m t h e cell s u s p e n s i o n w e r e first c o m p l e t e d a n d t h e n u m b e r of i m m a t u r e e r y t h r o i d cells w a s c a l c u l a t e d . T h e p r e p a r a t i o n of cell p a r t i c l e s of t h e size of m i t o c h o n d r i a f r o m s p l e n i c cells w a s c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d of L~WERL T h e m i t o c h o n d r i a or t h e p a r t i c l e s w e r e u s e d for t h e e n z y m e a s s a y . A c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e w a s a s s a y e d b y t h e m o d i f i e d m e t h o d of GRANICK AND URAT~V~. A s s h o w n in T a b l e I, t h e d e c r e a s e d o x y g e n t e n s i o n r e s u l t e d in a n i n c r e a s e d a c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e i n t h e s p l e e n , b u t t h e a c t i v i t y in t h e l i v e r r e m a i n e d u n c h a n g e d . O n t h e o t h e r h a n d , D D C i n c r e a s e d t h e a c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e in t h e l i v e r w i t h o u t i n f l u e n c i n g t h a t in t h e s p l e e n . H y p e r TABLE I D I F F E R E N T RESPONSES OF H E P A T I C AND E R Y T H R O P O I E T I C O-AMINOLEVULINIC ACID SYNTHETASP2
&Aminolevulinic acid synthetase activity was assayed in a standard mixture containing glycine, 5o/,moles; 2-ketoglutarate, 3/,moles; phosphate buffer (pH 7.4), 5o/*nloles; MgCI,,, 4 pmoles; EDTA, 2 pmoles; pyridoxal phosphate, o. 4/~mole; coenzyme A, o.2 5/~mole; enzyme, o.6 ml anti water to give a final volume of i.o nil. Following incubation in air for 2 h at 3 8(', the reactions were stopped by adding I.O ml of ~-O//o trichloroacetic acid and 6-aminolevulinic acid was determined by the method of MAUZERALL AND GRANICKIa. Each value represents the average and standard error obtained from 3 experiments using pooled tissues from 5 mice in each experiment. Figures in parentheses indicate number of mice. Treatment
Time
d-Aminolevulinic acid synlhesis . . . . . . Liver . . . Spleen . mltmoles d-aminolevulinic acid formed per g liver
Relative number of immature erythroid cells in lhe splenic cells (°/°)
per i o 8 splenic cells
during 2-h incubation
Control (15) Hypoxia * Hypoxia Hypoxia Hypoxia + hemin** Polycythemia*** (15) Polycythemia + erythropoietin § Polycythemia + erythropoietin Polycythemia + erythropoietin DDC§ DDC DDC + heroin**
20.0
I 2 4 4
i~ 1.2
1. 4 2.0 1.8 1-3 2. 4
10. I ~
3.04 4.15 6.98 6.75 o
13.2 17.6 29.1 27.4 o
i day
(5)
12.o
2.23
2 days
(5)
lO.8
4.55
15.o
1.o8 2.21 :~ o.41 2.35 i 0.43 2.29 + 0.35
-9.9 ~ 1.4 lO.2 ~- 1.2 lO. 4 ~ 1.6
11.6 92.3 ~ i2. 5 148.o ± 21. 5 76.5 @ 11.2
~ 0.32 _~ 0.36 -2 0.72 ~ 0.83
I. 5
18.2 2o.8 17. 9 16.8 lO.8
days (5) h (15) h (15) h (15)
@ + ~ ! k
2 . 3 1 @ O.21
(15) (15) (15) (15)
4 i2 24 24
day days days days
@ 2.1 :}: 2.8 ± 4.3 :~ 5.1
* Mice were kept in the hypoxic chamber (io vol. ~o Oo) for 12 h a day. ** Hemin solution was prepared by the method of SILVERSTEIN14 and injected (5o mg/kg) intraperitoneally every 2 4 h. ** * Mice were rendered polycythemic (hematocrit ;> 7° }~) by the transfusion nlethod 11. § A single dose of urinary erythropoietin (3oo CO units/kg) or DDC (5o0 nlg/kg) was injected subcutaneously at the beginning of the experiments. Biochim. Biophys. Acta, 148 (1967) 585-587
587
SHORT COMMUNICATIONS
transfusion, however, repressed the activity in the liver to approximately half the control and rendered the activity in the spleen undetectable. On the administration of erythropoietin to the polycythemic mice, the activity of 8-aminolevulinic acid synthetase in the liver remained decreased, while the activity in the spleen showed a gradual increase. This effect on the spleen was in accordance with the morphological observations of FILMANOWICZ AND GURNEY n a n d NAKAO et al. 12 who reported the gradual increase of erythroid cells in the spleen after erythropoietin injection to polycythemic mice in which erythroid cells had completely disappeared as a result of hypertransfusion. Heroin administration seems to inhibit partially the increase in the activity of hepatic 8-aminolevulinic acid synthetase in DDC-poisoned mice without influencing the increasing activity of splenic 8-aminolevulinic acid synthetase in the hypoxic mice. The decreased activity of 8-aminolevulinic acid synthetase in the liver of hypertransfused mice could be explained reasonably by the inhibitory effect of the excessive hemin formed from the injected erythrocytes in the liver. As shown in Table I, the levels of 8-aminolevulinic acid synthetase activity in the spleen were uniformly parallel to the relative numbers of immature erythroid cells in the spleen. These results suggest that the regulatory mechanism of 8-aminolevulinic acid synthetase activity might be different in the liver and the erythropoietic organ (spleen) in mice. Oxygen tension in blood might play an important role in the regulation of 8-aminolevulinic acid synthesis through the generation of new erythroid cells evoked by an action of erythropoietin in the erythropoietic organ, while in the liver other mechanisms, probably concerning the heine metabolism, might be involved in the regulation of 8-aminolevulinic acid synthesis. This work was supported in part by National Institutes of Health Research grant No. ROI-AM-o6454.
The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo (Japan)
OSAMU WADA SHIGERU SASSA FUMIMARO TAKAKU
Yuzo YANO GUMPEI URATA K I K U NAKAO
i 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. LASCELLES, Biochem. J., 72 (1959) 5o8. T. G. LBSSlE AND W. t{. SISTRON, Biochim. Biophys. Acta, 86 (1964) 25o. S. GRANICK AND G. URATA, J. Biol. Chem., 238 (1963) 821. K. ]2). GIBSON, W. G. LAVER AND A. NEUBERGER, Biochem. J., 7° (1958) 71. J. ONISAWA AND R. F. LABBE, Science, 14o (1963) 1326. J. LASCELLES, J. Gen. Microbiol., 23 (196o) 483. S. GRANICK, J. Biol. Chem., 241 (1966) 1359. A. D. WAXMAN, A. COLLINS AND ]2). P. TSCttUDY, Biochem. Biophys. Res. Commun., 24 (I966) 675. I. BRODSKY, L. H. DENNIS, S. B. KAHN AND L. W. BRADY, Cancer Res., 26 (1966) 198. G. H. HOGEBOOM AND W. C. SCHNEIDER, in S. P. COLOWICK AND N. O. KAPLAN, Methods of Enzymology, Vol. I, Academic Press, New York, I955, p. 16. E. FILMANOWICZ AND C. GURNEY, J. Lab. Clin. Med., 57 (1961) 65. I{. 7NAKAO, F. TAKAKU, S. FUJIOKA AND S. SASSA, Blood, 27 (1966) 537" D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 219 (1956) 435. E. SILVERSTEIN, Biochem. Pharmacol., i i (1962) 431.
Received June 26th, 1967 Revised manuscript received September Ist, 1967 Biochim. Biophys. Acta, 148 (1967) 585-587
585
will probably be most useful in studying conformational changes in synthetic polypeptides where only one reactive functional group is present. We are pleased to thank Professors S. A. BERNHARD and A. R. LARRABEE for helpful discussions and to National Science Foundation (Grant GP-55o9) for support of this work. D.L.N. was a National Science Foundation undergraduate research participant, summer 1965 .
Department of Chemistry, University of Oregon, Eugene, Oreg. 97403 (U.S.A.)
O. H . G R I F F I T H J. F. W. KEANA
D. L. NOALL J. L. IVEY
I T. J. STONE,T. BUCKMAN,13. L. NORDIO AND H. M. MCCONNELL, Proc. Natl. Acad. Sci. U.S., 54 (1965) iOlO. 2 L. STRYER ANn O. H. GRIFFITH, Proc. Natl. Acad. Sci. U.S., 54 (1965) 1785. 3 A. S. WAGGONER,O. H. GRIFFITH AND C. R. CHRISTENSEN, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1198. 4 S. OGAWA AND H. M. MCCONNELL, Proc. Natl. Acad. Sci. U.S., 58 (1967) 19. 5 E. G. ROZANTZI~V AND L. A. KRINITZKAYA, Tetrahedron, 21 (1965) 491. 6 D. S. TARBELL AND N. A. LglSTER, J. Org. Chem., 23 (1958) 1149. 7 T. B. WINDHOLZ, J. Org. Chem., 23 (1958) 2044. 8 T. B. WINDI~OLZ, J. Org. Chem., 25 (196o) 17o 3. 9 D. S. TARBlgLL AND E. J. LONGOSZ, J. Org. Chem., 24 (1959) 774. i o A. G. GORNALL, C. J. BARDAWILL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. i i N. A. LEISTER AND D. S. TARBELL, J. Org. Chem., 23 (1958) 1152.
Received August I8th, i967 Biochim. Biophys. Acta, 148 (1967) 583-585
BBA
2337o
Different responses of the hepatic and erythropoietic ~i-aminolevulinic acid
synthetase of mice ~-Aminolevulinie acid synthetase demonstrated in some species of microorganisms1, 2 and also in the livez a and erythropoietic organs 4 of mammals may be regarded as the rate-limiting enzyme in porphyrin and heine biosynthesis. Furthermore, a number of indirect observations 5 has suggested that the state of cellular oxidation might influence the metabolic regulation of porphyrin and heme biosynthesis through the activity of S-aminolevulinic acid synthetase. In microorganisms in fact, ~-aminolevulinic acid synthetase activity is shown to be regulated by oxygen and heme concentrations in the environment1,2,6. Furthermore, it has been shown by GRANICK~ with chick embryo liver cells and more recently by TSCHUDYs in rats, that hemin acts as a corepressor to prevent the formation of ~-aminolevulinic acid synthetase in the liver. The present experiments were designed to study the responses of the 3-aminolevulinic acid synthetase activity in the liver and erythropoietic organ of male mice (D-D strain, weighing 18-2o g) to hypoxia, polycythemia, erythropoietin and 3,5-dicarbethoxy-i,4-dihydroeollidine (DDC) as a porphyrinogenic substance. As the erythropoietic organ, we chose the spleen which has been shown to be analogous A b b r e v i a t i o n : DDC, 3 , 5 - d i c a r b e t h o x y - I , 4 - d i h y d r o c o l l i d i n e .
Biochim. Biophys. Acta, 148 (I967) 585-587
5~6
SHORT COMMUNICATIONS
t o b o n e m a r r o w f r o m t h e s t a n d p o i n t of e r y t h r o p o i e s i s i n m i c e 9. L i v e r m i t o c h o n d r i a w e r e p r e p a r e d a c c o r d i n g t o t h e m e t h o d of HOGEBOOM AaD SCHNEIDER ~°. F o r t h e p r e p a r a t i o n of m i t o c h o n d r i a f r o m s p l e n i c cells, t h e s p l e e n s o b t a i n e d f r o m 5 m i c e w e r e c r u s h e d a n d s u s p e n d e d i n 5 m l of c o o l e d i s o l o g o u s p l a s m a a n d p a s s e d t h r o u g h a fine m e s h . C o u n t s of n u c l e a t e d cells a n d r e t i c u l o c y t e s , a n d p r e p a r a t i o n of s m e a r s f r o m t h e cell s u s p e n s i o n w e r e first c o m p l e t e d a n d t h e n u m b e r of i m m a t u r e e r y t h r o i d cells w a s c a l c u l a t e d . T h e p r e p a r a t i o n of cell p a r t i c l e s of t h e size of m i t o c h o n d r i a f r o m s p l e n i c cells w a s c a r r i e d o u t a c c o r d i n g t o t h e m e t h o d of L~WERL T h e m i t o c h o n d r i a or t h e p a r t i c l e s w e r e u s e d for t h e e n z y m e a s s a y . A c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e w a s a s s a y e d b y t h e m o d i f i e d m e t h o d of GRANICK AND URAT~V~. A s s h o w n in T a b l e I, t h e d e c r e a s e d o x y g e n t e n s i o n r e s u l t e d in a n i n c r e a s e d a c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e i n t h e s p l e e n , b u t t h e a c t i v i t y in t h e l i v e r r e m a i n e d u n c h a n g e d . O n t h e o t h e r h a n d , D D C i n c r e a s e d t h e a c t i v i t y of 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e in t h e l i v e r w i t h o u t i n f l u e n c i n g t h a t in t h e s p l e e n . H y p e r TABLE I D I F F E R E N T RESPONSES OF H E P A T I C AND E R Y T H R O P O I E T I C O-AMINOLEVULINIC ACID SYNTHETASP2
&Aminolevulinic acid synthetase activity was assayed in a standard mixture containing glycine, 5o/,moles; 2-ketoglutarate, 3/,moles; phosphate buffer (pH 7.4), 5o/*nloles; MgCI,,, 4 pmoles; EDTA, 2 pmoles; pyridoxal phosphate, o. 4/~mole; coenzyme A, o.2 5/~mole; enzyme, o.6 ml anti water to give a final volume of i.o nil. Following incubation in air for 2 h at 3 8(', the reactions were stopped by adding I.O ml of ~-O//o trichloroacetic acid and 6-aminolevulinic acid was determined by the method of MAUZERALL AND GRANICKIa. Each value represents the average and standard error obtained from 3 experiments using pooled tissues from 5 mice in each experiment. Figures in parentheses indicate number of mice. Treatment
Time
d-Aminolevulinic acid synlhesis . . . . . . Liver . . . Spleen . mltmoles d-aminolevulinic acid formed per g liver
Relative number of immature erythroid cells in lhe splenic cells (°/°)
per i o 8 splenic cells
during 2-h incubation
Control (15) Hypoxia * Hypoxia Hypoxia Hypoxia + hemin** Polycythemia*** (15) Polycythemia + erythropoietin § Polycythemia + erythropoietin Polycythemia + erythropoietin DDC§ DDC DDC + heroin**
20.0
I 2 4 4
i~ 1.2
1. 4 2.0 1.8 1-3 2. 4
10. I ~
3.04 4.15 6.98 6.75 o
13.2 17.6 29.1 27.4 o
i day
(5)
12.o
2.23
2 days
(5)
lO.8
4.55
15.o
1.o8 2.21 :~ o.41 2.35 i 0.43 2.29 + 0.35
-9.9 ~ 1.4 lO.2 ~- 1.2 lO. 4 ~ 1.6
11.6 92.3 ~ i2. 5 148.o ± 21. 5 76.5 @ 11.2
~ 0.32 _~ 0.36 -2 0.72 ~ 0.83
I. 5
18.2 2o.8 17. 9 16.8 lO.8
days (5) h (15) h (15) h (15)
@ + ~ ! k
2 . 3 1 @ O.21
(15) (15) (15) (15)
4 i2 24 24
day days days days
@ 2.1 :}: 2.8 ± 4.3 :~ 5.1
* Mice were kept in the hypoxic chamber (io vol. ~o Oo) for 12 h a day. ** Hemin solution was prepared by the method of SILVERSTEIN14 and injected (5o mg/kg) intraperitoneally every 2 4 h. ** * Mice were rendered polycythemic (hematocrit ;> 7° }~) by the transfusion nlethod 11. § A single dose of urinary erythropoietin (3oo CO units/kg) or DDC (5o0 nlg/kg) was injected subcutaneously at the beginning of the experiments. Biochim. Biophys. Acta, 148 (1967) 585-587
587
SHORT COMMUNICATIONS
transfusion, however, repressed the activity in the liver to approximately half the control and rendered the activity in the spleen undetectable. On the administration of erythropoietin to the polycythemic mice, the activity of 8-aminolevulinic acid synthetase in the liver remained decreased, while the activity in the spleen showed a gradual increase. This effect on the spleen was in accordance with the morphological observations of FILMANOWICZ AND GURNEY n a n d NAKAO et al. 12 who reported the gradual increase of erythroid cells in the spleen after erythropoietin injection to polycythemic mice in which erythroid cells had completely disappeared as a result of hypertransfusion. Heroin administration seems to inhibit partially the increase in the activity of hepatic 8-aminolevulinic acid synthetase in DDC-poisoned mice without influencing the increasing activity of splenic 8-aminolevulinic acid synthetase in the hypoxic mice. The decreased activity of 8-aminolevulinic acid synthetase in the liver of hypertransfused mice could be explained reasonably by the inhibitory effect of the excessive hemin formed from the injected erythrocytes in the liver. As shown in Table I, the levels of 8-aminolevulinic acid synthetase activity in the spleen were uniformly parallel to the relative numbers of immature erythroid cells in the spleen. These results suggest that the regulatory mechanism of 8-aminolevulinic acid synthetase activity might be different in the liver and the erythropoietic organ (spleen) in mice. Oxygen tension in blood might play an important role in the regulation of 8-aminolevulinic acid synthesis through the generation of new erythroid cells evoked by an action of erythropoietin in the erythropoietic organ, while in the liver other mechanisms, probably concerning the heine metabolism, might be involved in the regulation of 8-aminolevulinic acid synthesis. This work was supported in part by National Institutes of Health Research grant No. ROI-AM-o6454.
The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo (Japan)
OSAMU WADA SHIGERU SASSA FUMIMARO TAKAKU
Yuzo YANO GUMPEI URATA K I K U NAKAO
i 2 3 4 5 6 7 8 9 IO ii 12 13 14
J. LASCELLES, Biochem. J., 72 (1959) 5o8. T. G. LBSSlE AND W. t{. SISTRON, Biochim. Biophys. Acta, 86 (1964) 25o. S. GRANICK AND G. URATA, J. Biol. Chem., 238 (1963) 821. K. ]2). GIBSON, W. G. LAVER AND A. NEUBERGER, Biochem. J., 7° (1958) 71. J. ONISAWA AND R. F. LABBE, Science, 14o (1963) 1326. J. LASCELLES, J. Gen. Microbiol., 23 (196o) 483. S. GRANICK, J. Biol. Chem., 241 (1966) 1359. A. D. WAXMAN, A. COLLINS AND ]2). P. TSCttUDY, Biochem. Biophys. Res. Commun., 24 (I966) 675. I. BRODSKY, L. H. DENNIS, S. B. KAHN AND L. W. BRADY, Cancer Res., 26 (1966) 198. G. H. HOGEBOOM AND W. C. SCHNEIDER, in S. P. COLOWICK AND N. O. KAPLAN, Methods of Enzymology, Vol. I, Academic Press, New York, I955, p. 16. E. FILMANOWICZ AND C. GURNEY, J. Lab. Clin. Med., 57 (1961) 65. I{. 7NAKAO, F. TAKAKU, S. FUJIOKA AND S. SASSA, Blood, 27 (1966) 537" D. MAUZERALL AND S. GRANICK, J. Biol. Chem., 219 (1956) 435. E. SILVERSTEIN, Biochem. Pharmacol., i i (1962) 431.
Received June 26th, 1967 Revised manuscript received September Ist, 1967 Biochim. Biophys. Acta, 148 (1967) 585-587