15. 7. 1975
Specialia
775
(Na + + K+)-ATPase stimulation (%) and % desmosterol of total sterols of erythroeyte ghosts (n = 8) A
B
C
D
Stimulation of .~[g++-ATPaseby Na + and K+ (%) P
113.1 (87.7-149.5)
92.3 (64.6-118.8)
50.2 (44.9-54.1)
44.5 (39.5-49.1)
Desmosterol of total sterols (%} P
75.4 (57-90)
~ 0.005
< 0.001 61.6 (43-75)
12.8 (11-15)
m
< 0.005
For the meaning of ABC and D see legend of Figure 1. P was determined by using the paired Student t-test.
A n d t h e m a i n t e n a n c e of a fluid-like e n v i r o n m e n t due t o its lipid c o n s t i t u e n t s is t h e p r e r e q u i s i t e for a n o p t i m a I f u n c t i o n of t r a n s p o r t e n z y m e s , as could be s h o w n b y spin label s t u d i e s 13, x4
13 j. SEELIG and W. HASSELBACH, Eur. J. Biochem. 21, 17 (1971). 14 C. M. GRISHAMand R. E. BARNETT, Biochemistry 12, 2635 (1973). 15 We are indebted to I. GELDMACHERand H. GOTTIG for skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinsehaft, Bonn-Bad Godesberg, and the Sonderforschungsbereich 90 of the University of Heidelberg.
Summary. Cholesterol of red blood cells (RBC) is readily e x c h a n g e d b y d e s m o s t e r o l a n d vice versa. The r e s u l t i n g a l t e r a t i o n in t h e sterol c o m p o s i t i o n influences t h e specific (Na + + K + ) - A T P a s e a c t i v i t y . I t is s u g g e s t e d t h a t t h i s effect is due t o a n a l t e r e d m e m b r a n e fluidity. W. FIEHN a n d D. SELLER 15
Medizinische Poliklinik der Universitiit, Hospitalstrasse 3, D-69 Heidelberg (German Federal Republic, BRD), 6 March 1975.
M o n o a m i n e Oxidase Activity in Helix pomatia I t has been r e p o r t e d t h a t a l t h o u g h m o n o a m i n e oxidase (MAO) exists in m o l l u s c a n n e r v o u s tissue, it is n o t able to m e t a b o l i z e a t r y p t a m i n e or s e r o t o n i n s u b s t r a t e 1. H o w ever, a n u m b e r of o t h e r e x p e r i m e n t s 2-7 h a v e s h o w n b o t h 5 - h y d r o x y t r y p t a m i n e (5-HT) a n d d o p a m i n e (DA) to be slightly m e t a b o l i z e d b y p r e s u m a b l y MAO, t h o u g h t h e a c t i v i t y is v e r y low. I t was t h e r e f o r e decided to a n a l y z e t h e d i s t r i b u t i o n a n d n a t u r e of MAO in t h e snail Helix pomatia in o r d e r to establish t h e f u n c t i o n a l significance of t h e e n z y m e in t h e n e r v o u s s y s t e m . Methods and materials. Tissues were d i s s e c t e d f r o m active snails into ice cold snail saline s, weighed a n d t h e n h o m o g e n i z e d in ice cold 0.15 WI t(C1, giving 10-100 nag tissue/nil. A l i q u o t s were t a k e n for p r o t e i n a s s a y 9. MAO a c t i v i t y was a n a l y z e d a c c o r d i n g to W u r t m a n a n d Axelrod ~0 w i t h m i n o r v a r i a t i o n s . The r e a c t i o n m i x t u r e h a d a t o t a l v o l u m e of 60 ~xl consisting of 20 [zl h o m o g e n a t e , 20 ~zl p h o s p h a t e buffer ( p H 7.4, 0.5 M), a n d 20 ~xl ~4Ct r y p t a m i n e solution (47 m C i / n m m l e ; 0.05 ~xCi d i l u t e d w i t h unlabelled t r y p t a m i n e to give 12 nmole/20 ~1 in 0.01 N HC1). The m i x t u r e was i n c u b a t e d a t 37~ for 60 min, s t o p p e d b y t h e a d d i t i o n of 70 ~xl 2 N tiC1 a n d 100 ~zl t h e r e f r o m twice e x t r a c t e d w i t h 2 m l toluene. The t o l u e n e was s e p a r a t e d f r o m t h e a q u e o u s p h a s e b y c e n t r i f u g a t i o n , a d d e d to 10 ml scintillation fluid a n d c o u n t e d in a P a c k a r d L i q u i d Scintillation Counter. 2 to 3 d e t e r m i n a t i o n s were m a d e f r o m each tissue h o m o g e n a t e . B l a n k values were o b t a i n e d as zero-time reactions. The a c t i v i t y was c o r r e c t e d for t h e efficiency of t h e e x t r a c t i o n p r o c e d u r e a n d exp r e s s e d as n m o l e t r y p t a m i n e m e t a b o l i z e d / g t i s s u e / m i n and nmole t r y p t a m i n e metabolized/g protein/min. I n several cases, 3H- 5-HT (17.3 Ci/mmole, 0.2 [zCi d i l u t e d w i t h unlabelled 5-HT t o give 6 nmole/20 ~zl):, 3H-DA (7.2 Ci/mmole, 0.1 ~Ci d i l u t e d to give 6 nmole/20 [zl) or ~4C-tryptamine (47 m C i / m n m l e , 0.1 ~xCi d i l u t e d to give 6 nmole/20 ~xl) was used as s u b s t r a t e a n d t h e resulting
p r o d u c t s e x t r a c t e d into 2 m l of e t h y l a c e t a t e / b e n z e n e (1 : 1 b y volume). To t e s t d r u g effects, d i s s e c t e d s u b o e s o p h a g e a l ganglia were p a r t i a l l y d e s h e a t e d a n d i n c u b a t e d for 60 rain a t 20 ~ in snail saline c o n t a i n i n g various c o n c e n t r a t i o n s of d r u g s t o g e t h e r w i t h 0.1 m g / m l ascorbic acid. The ganglia were t h e n b l o t t e d d r y a n d t h e i r MAO a c t i v i t y was analyzed. Results and discussion. A g r e a t v a r i a t i o n of MAO activi t y occurred in t h e d i f f e r e n t tissues (see Table), b e i n g h i g h e s t in t h e liver (62.11 n m o l e / g protein/rain) a n d a b s e n t in t h e a l b u m e n gland, flagella a n d r a d u l a r e t r a c t o r muscle. I n t h e n e r v o u s tissue, t h e MAO a c t i v i t y (nmole/g p r o t e i n / min) in t h e buccal ganglion was 26.81, while in t h e suprao e s o p h a g e a l a n d t h e s u b o e s o p h a g e a l ganglia it was 16.86 a n d 13.34 respectively. This is less t h a n 1% of t h a t r e p o r t ed for v e r t e b r a t e n e r v o u s tissue 11, b u t is similar t o t h a t f o u n d in o t h e r molluscs 12.
1 J . CARDOT, C. r. hebd. S6anc. Acad. Sci., Paris 258, 1103 (1964). 2 g. HIRIPI, Ann. Biol. Tihany 37, 33 (1970). 3 C. A. 1V[ARSDEN,Comp. gen. Pharmae. 3, 1 (1972). 4 N. N. OSBORNE, Dr. J. Pharlnae. 49, 170 (1973). 5 N. N. OSBORNE, E. PRIGGEMEIER and V. NEUttOFF, Experientia 29, 1351 (1973). e N. N. OSBORNE, E. PRIGGEMmER and V. NEUttOFF, Brain Res. 90, 261 (1975). 7 N. N. OSBORNE and G. A. COTTRELL, Conlp. gen. Pharmae. 1, 1 (1970). 8 K. MENG, Zool. Fahr. 68, 193 (1960). 90. H. LOWRY, N. J. ROSEBROUGH,A. L. FARRand R. J. RANDALL, J. bioh Chem. 793, 265 (1951). 10 R. J. WURTMANand J. AXEt, ROD, Biochem. Pharmac. 12, 1439 (1963). n F. M. ACHEE, G. TOGULAand S. GABAY,J. Neuroehem. 22, 651 (1974). 12 L. HIRIPI and J. SALANKI,Ann. Biol. Tihany 38, 31 (1971).
776
Specialia
EXPERIEN'TIA31/7
MAO a c t i v i t y p r o v e d t o be linear for a m i n i m u m of 60 m i n a t t e m p e r a t u r e s b e t w e e n 20~176 The Q10 (27~176 was f o u n d t o be 1.9. A t 0~ no a c t i v i t y was found, w h i c h is i n t e r e s t i n g in view of t h e f a c t t h a t snails can survive for m o n t h s a t t h i s t e m p e r a t u r e . This, t o g e t h e r w i t h o t h e r d a t a 1~, s h o w i n g a n u p t a k e m e c h a n i s m for 5 - H T t o e x i s t a t 0 ~ s u p p o r t s t h e idea t h a t MAO p l a y s o n l y a m i n o r role in t h e i n a c t i v a t i o n of n e u r o t r a n s m i t t e r s in t h e snail. MAO p r o p e r t i e s were s t u d i e d in m o r e d e t a i l in t h e s u b o e s o p h a g e a l ganglia. T h e Km v a l u e for t r y p t a m i n e was 2.1 • 10 -~ M, similar t o t h a t f o u n d in t h e r a b b i t b r a i n of 1 . 6 • -5 M n. U s i n g 5-HT a n d D A as s u b s t r a t e s , r a t h e r t h a n t r y p t a m i n e , it was f o u n d t h a t 5 - H T w a s d e a m i n a t e d a t a r a t e of only 20% a n d D A o n l y 50% of t h a t of t r y p t a m i n e . This c o n t r a s t s w i t h s t u d i e s in t h e v e r t e b r a t e s w h e r e h i g h e r specific activities were f o u n d for 5-HT a n d D A s u b s t r a t e s in c o n t r a s t t o t r y p t a m i n e n. The effects of v a r i o u s d r u g s on MAO a c t i v i t y were also studied. N i M a m i d e (10 -4 M), a MAO inhibitor, p r o d u c e d a 50 % decrease of MAO a c t i v i t y . C h l o r p r o m a z i n e (10-4 M), a D A r e c e p t o r blocker, also i n h i b i t e d MAO b y 5 0 % : C o n c e n t r a t i o n s of less t h a n 5 • 10 -5 M of e i t h e r d r u g h a d no m e a s u r a b l e effect. R e s e r p i n e (5 • 10 -5 M or greater) e n h a n c e d t h e MAO a c t i v i t y , w i t h a 50% increase a t 10 -4 M . I n c u b a t i o n of ganglia w i t h 5-HT (10-~ M - 10 -3 M ) r e s u l t e d in a n o n - l i n e a r decrease in m e a s u r e d a c t i v i t y , t h o u g h t h i s m i g h t be due t o c o m p e t i t i o n of t h e a c c u m u l a t e d 5-HT w i t h t h e l~C-tryptamine. The effects of p a r g y l i n e (MAO inhibitor) were also e x a m i n e d . 5-I-IT d e a m i n a t i o n was o n l y i n h i b i t e d w i t h pargyline concentrations greater than 5 • -4 M . D A a n d t r y p t a m i n e d e a m i n a t i o n were b o t h p a r t i a l l y i n h i b i t e d
b y p a r g y l i n e b e t w e e n 5 • 10 -4 M a n d 5 • 10 -5 M . L i t t l e effect w a s seen a t c o n c e n t r a t i o n s lower t h a n 10 -~ M . This is i n t e r e s t i n g in v i e w of t h e f a c t t h a t m u l t i p l e f o r m s of MAO h a v e b e e n proposedl4-1* to occur in t h e v e r t e b r a t e s , b a s e d on t h e d i f f e r e n t i a l d e g r a d a t i o n of v a r i o u s m o n o a m i n e s a n d t h e a c t u a l s e p a r a t i o n of ' i s o e n z y m e s ' f r o m p u r i f i e d p r e p a r a t i o n s . The suggestion has b e e n p u t f o r w a r d t h a t p a r g y l i n e (MAO inhibitor) selectively i n h i b i t s t h e ]3 t y p e e n z y m e 17 ( s u b s t r a t e t y r a m i n e ) a t low conc e n t r a t i o n s , while t h e A t y p e e n z y m e (where 5 - H T is a s u b s t r a t e ) is only i n h i b i t e d b y h i g h e r c o n c e n t r a t i o n s of pargyline. D A u n d t r y p t a m i n e (being) s u b s t r a t e s for b o t h t y p e s , are p a r t i a l l y i n h i b i t e d a t low c o n c e n t r a t i o n s . Such is f o u n d t o be t h e case in t h e snail. I t is t h e r e f o r e possible t h a t m o r e t h a n one f o r m of MAO occurs in t h e snail, t h o u g h t h e significance of t h i s is n o t clear, especially in v i e w of t h e a p p a r a n t m i n o r role MAO m i g h t h a v e in snails. The significance of t h e s m a l l b u t definite a c t i v i t y of MAO in t h e snail c e n t r a l n e r v o u s s y s t e m is n o t clear, especially since o t h e r s t u d i e s 13 h a v e s h o w n r e - u p t a k e of t h e released t r a n s m i t t e r t o be t h e p r o b a b l e m e c h a n i s m of i n a c t i v a t i o n of D A a n d 5-HT in t h e snail. I t m a y well b e t h a t MAO h a s a n i n a c t i v a t i n g f u n c t i o n in only a s m a l l p r o p o r t i o n of n e r v e cells, b u t t h e ' b a c k g r o u n d noise' of t h e o t h e r cells, caused w h e n a n a l y z i n g whole ganglia, blurs t h e d i s c o v e r y of t h i s f u n c t i o n a l significance. H o w ever, it could be t h a t MAO h a s an e v e n d i s t r i b u t i o n , as has b e e n s h o w n for o t h e r i n a c t i v a t i n g e n z y m e s (acetylcholine-esterase13 a n d c a t e c h o l - 0 - m e t h y l - t r a n s f e r a s e 1 3 ) . I n t h i s case, t h e e n z y m e could be f u n c t i o n i n g m o r e as a n extraneuronal regulating mechanism, guarding against non-specific s t i m u l a t i o n b y e x t r a n e o u s amines.
MAO activity in various tissues
Summary. The d i s t r i b u t i o n a n d c h a r a c t e r i z a t i o n of MAO in v a r i o u s tissues of t h e snail were analyzed. O n l y low a m o u n t s of t h e e n z y m e exist in t h e d i f f e r e n t tissues a n d d a t a suggest t h a t t h e r e is m o r e t h a n one t y p e of MAO. P. ]3. GUTHRIE, V. NEUHOFF a n d N. N. OSBORNE
Tissue
Tissue (nmole/g/min)
Protein (nmole/g/min)
Albumen Gland Flagella Radula retractor muscle Liver Kidney Salivary gland Spermatheea Optic tentacle Ventricle Ganglia Bueeal Supraesophageal Suboesophageal Suboesophagel 9 Suboesophagelb
ND ND ND 8.053 :t: 0.390 2.506 4- 0.282 3.065 4- 0.268 0.465 -[- 0.099 0.105 4- 0.015 0.067 :L 0.001
ND ND ND 62.11 4- 4.05 25.15 4- 2.81 39.09 • 3.23 33.41 4- 0.52 0.80 4- 0.15 0.99 :t: 0.08
3.253 J: 0.262 1.502 4- 0.103 0.904 4- 0.069 0.189 4- 0.069 b 0.415 4- 0.018 b
26.81 4- 4.30 16.86 -4- 1.07 13.34 ~_~0.81 2.63 4- 0.96 ~ 5.77 :k 0.25b
Tissues analyzed as described ill text with tryptamine as substrate except s with 5-HT as substrate and b with DA as substrate. ND indicates not detected. Values are 4- SEM for n between 9-37.
Max-Planck- Institut /i~r experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Strasse 3, D-3dO0 G6ttingen (German Federal Republic, BRD), 21 February 1975. 18 N. N. OSBORNE, L. HIRIPI and V. ~EUHOFF, Bioehem. Pharmac.,
in press. 14 R. F. SQUIRES, Biochenl. Pharmae. 17, 1401 (1968). 15 R. F. SQUIRES, iI1 Advances in Biochemical Psychopharmacology
(Eds. E. COSTAand M. SANDLER; Raven Press, New York 1972), voI. 5, p. 355. 1, N. H. NEFF, H.-Y. T. YANGand C. GORIDIS, in Frontiers in Catecholamine Research (Eds. E. USDIN and S. H. SNYDER; Pergamon Press, Oxford 1973), p. 133. 1Tj . p. JOHNSTON, Biochem. Pharmae. 17, 1285 (1968). is p. C. EMSO~ and F. FONIqUM, J. Neurochem. 22, 1079 (1974). 19 R. E. 1V~cCAMA~qand S. A: DEWHURST, J. Neurochem. 78, 1329 (1971).
Phospholipids of Mycobacterium phlei ~ A m o n g s t t h e p h o s p h o l i p i d s of m y c o b a c t e r i a , cardiolipin has b e e n r e p o r t e d to be t h e m a j o r c o m p o n e n t in M. phlei (penso)3, in a n unclassified m y c o b a c t e r i a P63 a n d also in I.i3:Rv, H3vRa a n d M. avium (all NCTC) s t r a i n s 4. R e c e n t l y CHANDRAMOULI a n d VENKITASUBRAMANIAN5 h a v e r e p o r t e d f r o m t h i s l a b o r a t o r y t h a t t h e cardiolipin is t h e m a j o r c o m p o n e n t in M. smegmatis a n d
1 This investigation was supported in part by a PL-480 grant No. FG-In-336. 2 y. AKAMATSUand S. NOJIMA, J. Biochem. (Japan) 57, 430 (1965). 3 M. 1V[OTOMIYA,A. I~AYAMA, IV[. FUJIMOTO, H. SATO and S. OKA, Chem. Phys. Lipids 3, 159 (1969). 4 D. SUBRAHMANYAM,Can. J. Biochem. d2, 1195 (1964). 5 V. CHANDRAMOULIand T. A. VENKITASUBRAMANIAIq,Indian. J. Chest. Dis. 76, 199 (1974).
Specialia
775
(Na + + K+)-ATPase stimulation (%) and % desmosterol of total sterols of erythroeyte ghosts (n = 8) A
B
C
D
Stimulation of .~[g++-ATPaseby Na + and K+ (%) P
113.1 (87.7-149.5)
92.3 (64.6-118.8)
50.2 (44.9-54.1)
44.5 (39.5-49.1)
Desmosterol of total sterols (%} P
75.4 (57-90)
~ 0.005
< 0.001 61.6 (43-75)
12.8 (11-15)
m
< 0.005
For the meaning of ABC and D see legend of Figure 1. P was determined by using the paired Student t-test.
A n d t h e m a i n t e n a n c e of a fluid-like e n v i r o n m e n t due t o its lipid c o n s t i t u e n t s is t h e p r e r e q u i s i t e for a n o p t i m a I f u n c t i o n of t r a n s p o r t e n z y m e s , as could be s h o w n b y spin label s t u d i e s 13, x4
13 j. SEELIG and W. HASSELBACH, Eur. J. Biochem. 21, 17 (1971). 14 C. M. GRISHAMand R. E. BARNETT, Biochemistry 12, 2635 (1973). 15 We are indebted to I. GELDMACHERand H. GOTTIG for skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinsehaft, Bonn-Bad Godesberg, and the Sonderforschungsbereich 90 of the University of Heidelberg.
Summary. Cholesterol of red blood cells (RBC) is readily e x c h a n g e d b y d e s m o s t e r o l a n d vice versa. The r e s u l t i n g a l t e r a t i o n in t h e sterol c o m p o s i t i o n influences t h e specific (Na + + K + ) - A T P a s e a c t i v i t y . I t is s u g g e s t e d t h a t t h i s effect is due t o a n a l t e r e d m e m b r a n e fluidity. W. FIEHN a n d D. SELLER 15
Medizinische Poliklinik der Universitiit, Hospitalstrasse 3, D-69 Heidelberg (German Federal Republic, BRD), 6 March 1975.
M o n o a m i n e Oxidase Activity in Helix pomatia I t has been r e p o r t e d t h a t a l t h o u g h m o n o a m i n e oxidase (MAO) exists in m o l l u s c a n n e r v o u s tissue, it is n o t able to m e t a b o l i z e a t r y p t a m i n e or s e r o t o n i n s u b s t r a t e 1. H o w ever, a n u m b e r of o t h e r e x p e r i m e n t s 2-7 h a v e s h o w n b o t h 5 - h y d r o x y t r y p t a m i n e (5-HT) a n d d o p a m i n e (DA) to be slightly m e t a b o l i z e d b y p r e s u m a b l y MAO, t h o u g h t h e a c t i v i t y is v e r y low. I t was t h e r e f o r e decided to a n a l y z e t h e d i s t r i b u t i o n a n d n a t u r e of MAO in t h e snail Helix pomatia in o r d e r to establish t h e f u n c t i o n a l significance of t h e e n z y m e in t h e n e r v o u s s y s t e m . Methods and materials. Tissues were d i s s e c t e d f r o m active snails into ice cold snail saline s, weighed a n d t h e n h o m o g e n i z e d in ice cold 0.15 WI t(C1, giving 10-100 nag tissue/nil. A l i q u o t s were t a k e n for p r o t e i n a s s a y 9. MAO a c t i v i t y was a n a l y z e d a c c o r d i n g to W u r t m a n a n d Axelrod ~0 w i t h m i n o r v a r i a t i o n s . The r e a c t i o n m i x t u r e h a d a t o t a l v o l u m e of 60 ~xl consisting of 20 [zl h o m o g e n a t e , 20 ~zl p h o s p h a t e buffer ( p H 7.4, 0.5 M), a n d 20 ~xl ~4Ct r y p t a m i n e solution (47 m C i / n m m l e ; 0.05 ~xCi d i l u t e d w i t h unlabelled t r y p t a m i n e to give 12 nmole/20 ~1 in 0.01 N HC1). The m i x t u r e was i n c u b a t e d a t 37~ for 60 min, s t o p p e d b y t h e a d d i t i o n of 70 ~xl 2 N tiC1 a n d 100 ~zl t h e r e f r o m twice e x t r a c t e d w i t h 2 m l toluene. The t o l u e n e was s e p a r a t e d f r o m t h e a q u e o u s p h a s e b y c e n t r i f u g a t i o n , a d d e d to 10 ml scintillation fluid a n d c o u n t e d in a P a c k a r d L i q u i d Scintillation Counter. 2 to 3 d e t e r m i n a t i o n s were m a d e f r o m each tissue h o m o g e n a t e . B l a n k values were o b t a i n e d as zero-time reactions. The a c t i v i t y was c o r r e c t e d for t h e efficiency of t h e e x t r a c t i o n p r o c e d u r e a n d exp r e s s e d as n m o l e t r y p t a m i n e m e t a b o l i z e d / g t i s s u e / m i n and nmole t r y p t a m i n e metabolized/g protein/min. I n several cases, 3H- 5-HT (17.3 Ci/mmole, 0.2 [zCi d i l u t e d w i t h unlabelled 5-HT t o give 6 nmole/20 ~zl):, 3H-DA (7.2 Ci/mmole, 0.1 ~Ci d i l u t e d to give 6 nmole/20 [zl) or ~4C-tryptamine (47 m C i / m n m l e , 0.1 ~xCi d i l u t e d to give 6 nmole/20 ~xl) was used as s u b s t r a t e a n d t h e resulting
p r o d u c t s e x t r a c t e d into 2 m l of e t h y l a c e t a t e / b e n z e n e (1 : 1 b y volume). To t e s t d r u g effects, d i s s e c t e d s u b o e s o p h a g e a l ganglia were p a r t i a l l y d e s h e a t e d a n d i n c u b a t e d for 60 rain a t 20 ~ in snail saline c o n t a i n i n g various c o n c e n t r a t i o n s of d r u g s t o g e t h e r w i t h 0.1 m g / m l ascorbic acid. The ganglia were t h e n b l o t t e d d r y a n d t h e i r MAO a c t i v i t y was analyzed. Results and discussion. A g r e a t v a r i a t i o n of MAO activi t y occurred in t h e d i f f e r e n t tissues (see Table), b e i n g h i g h e s t in t h e liver (62.11 n m o l e / g protein/rain) a n d a b s e n t in t h e a l b u m e n gland, flagella a n d r a d u l a r e t r a c t o r muscle. I n t h e n e r v o u s tissue, t h e MAO a c t i v i t y (nmole/g p r o t e i n / min) in t h e buccal ganglion was 26.81, while in t h e suprao e s o p h a g e a l a n d t h e s u b o e s o p h a g e a l ganglia it was 16.86 a n d 13.34 respectively. This is less t h a n 1% of t h a t r e p o r t ed for v e r t e b r a t e n e r v o u s tissue 11, b u t is similar t o t h a t f o u n d in o t h e r molluscs 12.
1 J . CARDOT, C. r. hebd. S6anc. Acad. Sci., Paris 258, 1103 (1964). 2 g. HIRIPI, Ann. Biol. Tihany 37, 33 (1970). 3 C. A. 1V[ARSDEN,Comp. gen. Pharmae. 3, 1 (1972). 4 N. N. OSBORNE, Dr. J. Pharlnae. 49, 170 (1973). 5 N. N. OSBORNE, E. PRIGGEMEIER and V. NEUttOFF, Experientia 29, 1351 (1973). e N. N. OSBORNE, E. PRIGGEMmER and V. NEUttOFF, Brain Res. 90, 261 (1975). 7 N. N. OSBORNE and G. A. COTTRELL, Conlp. gen. Pharmae. 1, 1 (1970). 8 K. MENG, Zool. Fahr. 68, 193 (1960). 90. H. LOWRY, N. J. ROSEBROUGH,A. L. FARRand R. J. RANDALL, J. bioh Chem. 793, 265 (1951). 10 R. J. WURTMANand J. AXEt, ROD, Biochem. Pharmac. 12, 1439 (1963). n F. M. ACHEE, G. TOGULAand S. GABAY,J. Neuroehem. 22, 651 (1974). 12 L. HIRIPI and J. SALANKI,Ann. Biol. Tihany 38, 31 (1971).
776
Specialia
EXPERIEN'TIA31/7
MAO a c t i v i t y p r o v e d t o be linear for a m i n i m u m of 60 m i n a t t e m p e r a t u r e s b e t w e e n 20~176 The Q10 (27~176 was f o u n d t o be 1.9. A t 0~ no a c t i v i t y was found, w h i c h is i n t e r e s t i n g in view of t h e f a c t t h a t snails can survive for m o n t h s a t t h i s t e m p e r a t u r e . This, t o g e t h e r w i t h o t h e r d a t a 1~, s h o w i n g a n u p t a k e m e c h a n i s m for 5 - H T t o e x i s t a t 0 ~ s u p p o r t s t h e idea t h a t MAO p l a y s o n l y a m i n o r role in t h e i n a c t i v a t i o n of n e u r o t r a n s m i t t e r s in t h e snail. MAO p r o p e r t i e s were s t u d i e d in m o r e d e t a i l in t h e s u b o e s o p h a g e a l ganglia. T h e Km v a l u e for t r y p t a m i n e was 2.1 • 10 -~ M, similar t o t h a t f o u n d in t h e r a b b i t b r a i n of 1 . 6 • -5 M n. U s i n g 5-HT a n d D A as s u b s t r a t e s , r a t h e r t h a n t r y p t a m i n e , it was f o u n d t h a t 5 - H T w a s d e a m i n a t e d a t a r a t e of only 20% a n d D A o n l y 50% of t h a t of t r y p t a m i n e . This c o n t r a s t s w i t h s t u d i e s in t h e v e r t e b r a t e s w h e r e h i g h e r specific activities were f o u n d for 5-HT a n d D A s u b s t r a t e s in c o n t r a s t t o t r y p t a m i n e n. The effects of v a r i o u s d r u g s on MAO a c t i v i t y were also studied. N i M a m i d e (10 -4 M), a MAO inhibitor, p r o d u c e d a 50 % decrease of MAO a c t i v i t y . C h l o r p r o m a z i n e (10-4 M), a D A r e c e p t o r blocker, also i n h i b i t e d MAO b y 5 0 % : C o n c e n t r a t i o n s of less t h a n 5 • 10 -5 M of e i t h e r d r u g h a d no m e a s u r a b l e effect. R e s e r p i n e (5 • 10 -5 M or greater) e n h a n c e d t h e MAO a c t i v i t y , w i t h a 50% increase a t 10 -4 M . I n c u b a t i o n of ganglia w i t h 5-HT (10-~ M - 10 -3 M ) r e s u l t e d in a n o n - l i n e a r decrease in m e a s u r e d a c t i v i t y , t h o u g h t h i s m i g h t be due t o c o m p e t i t i o n of t h e a c c u m u l a t e d 5-HT w i t h t h e l~C-tryptamine. The effects of p a r g y l i n e (MAO inhibitor) were also e x a m i n e d . 5-I-IT d e a m i n a t i o n was o n l y i n h i b i t e d w i t h pargyline concentrations greater than 5 • -4 M . D A a n d t r y p t a m i n e d e a m i n a t i o n were b o t h p a r t i a l l y i n h i b i t e d
b y p a r g y l i n e b e t w e e n 5 • 10 -4 M a n d 5 • 10 -5 M . L i t t l e effect w a s seen a t c o n c e n t r a t i o n s lower t h a n 10 -~ M . This is i n t e r e s t i n g in v i e w of t h e f a c t t h a t m u l t i p l e f o r m s of MAO h a v e b e e n proposedl4-1* to occur in t h e v e r t e b r a t e s , b a s e d on t h e d i f f e r e n t i a l d e g r a d a t i o n of v a r i o u s m o n o a m i n e s a n d t h e a c t u a l s e p a r a t i o n of ' i s o e n z y m e s ' f r o m p u r i f i e d p r e p a r a t i o n s . The suggestion has b e e n p u t f o r w a r d t h a t p a r g y l i n e (MAO inhibitor) selectively i n h i b i t s t h e ]3 t y p e e n z y m e 17 ( s u b s t r a t e t y r a m i n e ) a t low conc e n t r a t i o n s , while t h e A t y p e e n z y m e (where 5 - H T is a s u b s t r a t e ) is only i n h i b i t e d b y h i g h e r c o n c e n t r a t i o n s of pargyline. D A u n d t r y p t a m i n e (being) s u b s t r a t e s for b o t h t y p e s , are p a r t i a l l y i n h i b i t e d a t low c o n c e n t r a t i o n s . Such is f o u n d t o be t h e case in t h e snail. I t is t h e r e f o r e possible t h a t m o r e t h a n one f o r m of MAO occurs in t h e snail, t h o u g h t h e significance of t h i s is n o t clear, especially in v i e w of t h e a p p a r a n t m i n o r role MAO m i g h t h a v e in snails. The significance of t h e s m a l l b u t definite a c t i v i t y of MAO in t h e snail c e n t r a l n e r v o u s s y s t e m is n o t clear, especially since o t h e r s t u d i e s 13 h a v e s h o w n r e - u p t a k e of t h e released t r a n s m i t t e r t o be t h e p r o b a b l e m e c h a n i s m of i n a c t i v a t i o n of D A a n d 5-HT in t h e snail. I t m a y well b e t h a t MAO h a s a n i n a c t i v a t i n g f u n c t i o n in only a s m a l l p r o p o r t i o n of n e r v e cells, b u t t h e ' b a c k g r o u n d noise' of t h e o t h e r cells, caused w h e n a n a l y z i n g whole ganglia, blurs t h e d i s c o v e r y of t h i s f u n c t i o n a l significance. H o w ever, it could be t h a t MAO h a s an e v e n d i s t r i b u t i o n , as has b e e n s h o w n for o t h e r i n a c t i v a t i n g e n z y m e s (acetylcholine-esterase13 a n d c a t e c h o l - 0 - m e t h y l - t r a n s f e r a s e 1 3 ) . I n t h i s case, t h e e n z y m e could be f u n c t i o n i n g m o r e as a n extraneuronal regulating mechanism, guarding against non-specific s t i m u l a t i o n b y e x t r a n e o u s amines.
MAO activity in various tissues
Summary. The d i s t r i b u t i o n a n d c h a r a c t e r i z a t i o n of MAO in v a r i o u s tissues of t h e snail were analyzed. O n l y low a m o u n t s of t h e e n z y m e exist in t h e d i f f e r e n t tissues a n d d a t a suggest t h a t t h e r e is m o r e t h a n one t y p e of MAO. P. ]3. GUTHRIE, V. NEUHOFF a n d N. N. OSBORNE
Tissue
Tissue (nmole/g/min)
Protein (nmole/g/min)
Albumen Gland Flagella Radula retractor muscle Liver Kidney Salivary gland Spermatheea Optic tentacle Ventricle Ganglia Bueeal Supraesophageal Suboesophageal Suboesophagel 9 Suboesophagelb
ND ND ND 8.053 :t: 0.390 2.506 4- 0.282 3.065 4- 0.268 0.465 -[- 0.099 0.105 4- 0.015 0.067 :L 0.001
ND ND ND 62.11 4- 4.05 25.15 4- 2.81 39.09 • 3.23 33.41 4- 0.52 0.80 4- 0.15 0.99 :t: 0.08
3.253 J: 0.262 1.502 4- 0.103 0.904 4- 0.069 0.189 4- 0.069 b 0.415 4- 0.018 b
26.81 4- 4.30 16.86 -4- 1.07 13.34 ~_~0.81 2.63 4- 0.96 ~ 5.77 :k 0.25b
Tissues analyzed as described ill text with tryptamine as substrate except s with 5-HT as substrate and b with DA as substrate. ND indicates not detected. Values are 4- SEM for n between 9-37.
Max-Planck- Institut /i~r experimentelle Medizin, Forschungsstelle Neurochemie, Hermann-Rein-Strasse 3, D-3dO0 G6ttingen (German Federal Republic, BRD), 21 February 1975. 18 N. N. OSBORNE, L. HIRIPI and V. ~EUHOFF, Bioehem. Pharmac.,
in press. 14 R. F. SQUIRES, Biochenl. Pharmae. 17, 1401 (1968). 15 R. F. SQUIRES, iI1 Advances in Biochemical Psychopharmacology
(Eds. E. COSTAand M. SANDLER; Raven Press, New York 1972), voI. 5, p. 355. 1, N. H. NEFF, H.-Y. T. YANGand C. GORIDIS, in Frontiers in Catecholamine Research (Eds. E. USDIN and S. H. SNYDER; Pergamon Press, Oxford 1973), p. 133. 1Tj . p. JOHNSTON, Biochem. Pharmae. 17, 1285 (1968). is p. C. EMSO~ and F. FONIqUM, J. Neurochem. 22, 1079 (1974). 19 R. E. 1V~cCAMA~qand S. A: DEWHURST, J. Neurochem. 78, 1329 (1971).
Phospholipids of Mycobacterium phlei ~ A m o n g s t t h e p h o s p h o l i p i d s of m y c o b a c t e r i a , cardiolipin has b e e n r e p o r t e d to be t h e m a j o r c o m p o n e n t in M. phlei (penso)3, in a n unclassified m y c o b a c t e r i a P63 a n d also in I.i3:Rv, H3vRa a n d M. avium (all NCTC) s t r a i n s 4. R e c e n t l y CHANDRAMOULI a n d VENKITASUBRAMANIAN5 h a v e r e p o r t e d f r o m t h i s l a b o r a t o r y t h a t t h e cardiolipin is t h e m a j o r c o m p o n e n t in M. smegmatis a n d
1 This investigation was supported in part by a PL-480 grant No. FG-In-336. 2 y. AKAMATSUand S. NOJIMA, J. Biochem. (Japan) 57, 430 (1965). 3 M. 1V[OTOMIYA,A. I~AYAMA, IV[. FUJIMOTO, H. SATO and S. OKA, Chem. Phys. Lipids 3, 159 (1969). 4 D. SUBRAHMANYAM,Can. J. Biochem. d2, 1195 (1964). 5 V. CHANDRAMOULIand T. A. VENKITASUBRAMANIAIq,Indian. J. Chest. Dis. 76, 199 (1974).
