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Sugar derivatives of indolamines, Part II: Difference in the oxidation of serotonin and its desoxyfructose derivative by rat brain monoamine oxidase
SUGAR
DERlVATlVES
QXlDATlON
OF INDOLAlLiINES,
OF SEROTONIN
AND ITS
PART
RAT BRAIN MONOAhilNE V.R.Gujrati
K.P.Bhargava, King
Lnstitut
Medical
George’s
d2
Chimie
(Received
Dept.of
M.Mester
and
Substances
28.10.1977; Sccepted
by
: DIFFERENCE
IN THE
DERIVATIVE
BY
OXIDASE and
College,
des
II
DESOXYFRUCTOSE
Eashee
Pharmacology,
Xii, Lucknor,
India
L .Mester :Gaturelles,
in Editor
revised W.H.
Gif-Sur-‘Yvette
form
23.2.1978.
Seegers)
Desoxyfructo-serotonin is a new synthetic sugar derivative of sero tonin, however, its formation in physiological conditions can also be expected. Using rat brain monoamine oxidase (MAO) in vitro, desoxyfructo-serotonin is significantly more slowly metabolized than serotonin. “Transcypromine”, a specific inhibitor of MAO, also inhibits the oxidative deamination of the sugar derivative.
INTRODUCTION I-Desoxy-I-(5-hydroxytryptamino)-D-fructose is a new, stable, strongly reducing sugar derivative of serotonin, which was obtained by heating serotonin oxalate with D-glucose (1) in absolute ethanol. Since 1975, the formation of similar sugar derivatives in the blood has been suggested (2). Subsequently, the existence in the blood of the desoxysugar derivative of haemoglobin has been reported (3,4), but not yet of serotonin. Nevertheless, the sugar derivative could be a possible form of circulating serotonin. Desoxyfructo-serotonin, when compared with serotonin, shor
791
SEROTOXIN
792
X-iD FiONOAHIXE
OXIDASE
Vo1.12,No.5
XETHODS Albino rats were killed by decapitation, the brain was quickly excised and homogenized in ice-cold 0.25 X sucrose with a Potter-Elvehjem homogenizer. The tissue homogenate (10X, w/v) thus obtained was centrifuged in refrigerated centrifuge at 8OOxg for 30 minutes and the supernatant decanted. The residue (A) was rehomogenized in 0.25 M sucrose and again centrifuged. The washings were mi:ed with tEe supernatant. The supernatant so obtained, was again centrifuged at 16,OOOxg for 40 minutes and supernatant decanted. The residue (B) was washed twice 0.25 M sucrose and again centrifuged at I6,ODDxg. The residue (C) soobtained consisted of mitochondrial particles and was suspended in 0.25 g sucrose (10% w/v), for the assay of MAO activity. The assay system consisted to 1.0 ml of 0.25 M phosphate buffer (pH 7.4). Suitably diluted enzyme preparations and various concentrawere incubated at 37°C tions of the substrates (5-HT or DF-5HT) The reaction was stopped by addition of I ml of 10% Eor 30 min. The tubes were centrifugated at IOOOxg for trichloroacetic acid. assayed for S-HT by the 20 min. and the supernatant was calorimetric method (7). Inhibition experiments were done using “tranylcypromine” a specific MAO inhibitor, (8) (trans-2-Phenylcyclopropylamine), The reaction mixture in a final and DF-5HT as the substrate. volume of 2 ml consisted of DF-5-HT (final concentration 0.05 z), I ml of phosphate buffer (0.5 I), suitable amount of inhibitor and 0.3 ml purified rat brain mitochondrial preparation. and water, Enzyme with the inhibitor was preincubated for 15 min. at 37°C prior to the addition of the substrate and the reaction was Activity was determined stopped after 30 min incubation. Each experiment was done in duplicate and colotimetrically (7). the mean value of the duplicate. the values represented are
RESULTS
were
The carried
rat out
brain with
AND
DISCUSSION
MAO mediated oxidation purified mitochondrial
of
5-HT and preparation
DF-5HT (6).
The substrate saturation curves of 5-HT and DF-5HT obtained from various concentrations (0.05 to 0.5 mM), were hyperbolic, thus exhibiting classical Michaelis kinetics (Fig.1). When Lineweaver-Burk curves were plotted (substrate concentrations ranging from 0.83 to 6.66 mM), the Km value of MAO for DF-5HT M) vas almost twice that of 5-HT (8.0 x low3 H) (14.28 x iO-3 This confirms that the affinity of MAO for DF-5HT is (Fig.2). considerably less when compared with 5-HT in vitro. inhibits a specific MAO inhibitor “Tranylcypromine”, (8), the oxidative deamination of DF-5HT. The results given in TABLE I, shoving 35 and 65.7 % inhibition of the MAO activity, respectively, con1irms the degradation of the sugar derivative by the enzyme MAO (Table I).
i-01.
17
,so.
5
5-HT
0.0 -
0.6-
0.4-
DF-5-HT
0.05
0
mM
0.1
0.15
SUBSTRATE
Effect of 5-HT and DF-5HT activity expressed as
0.2
0.25
0.3
0.35
0.4
CONCENTRATION
FIG. 1 concentration on rat brain change in optical density
r
>fAO
PLOT
‘LINEWEAVER -6URK
I
-- 1 = Km
5-HT h&r)
25
i 0.07
Km -14.
OF-5 28x
6’~
0~125
KIII =&oo
x 10‘~
M
.
6.0-
_A
KIII
I/ [S] Y
IO-sM
FIG. 2 Lineweaver-Burk plot showing oxidative deamination of 5-HT and DF-SHT by purified rat brain mitochondrial preparation. Initial velocity (v) was expressed as change in optical density (O.D./ 30 min.). The substrate concentration (S) was expressed as l/(S) xIO-~ M.
HT
SEROTOSIN
794
XND
ElONO~hlINE OXIDASE
I
TABLE OXIDATION Name
of
Without
*
OF
OF-5HT
inhibitor
Vo1.12,No.J
BY TRANYLCYPROMINE Concentration
INHIBITED
Z Activity
of
MAO MAO
1oo*
inhibitor
Tranylcypromine
IO -6
y.
65.0
Tranylcypromine
IO -4 M
34.3
The activity at pH 7.4 of control MAO + OF-5HT was taken as 100 z. The values in the table represent the per cent activity in presence of tranylcypromine at two different concentrations.
readily
It is conceivable metabolized and
that in vivo also DF-5HT may not be exhibit prolonged serotonin-like effects.
CONCLUSION The observed difference in the rate of oxidation of desoxyfructo-serotonin and serotonin with monoamine oxidase is due probably to a decreased affinity of the sugar derivative towards If the formation of the sugar derivative occurs in the enzyme. it may effect the metabolism and the the blood, as suggested, mode of action of serotonin in haemostasis.
ACKNOWLEDGEMENT This research was French-Indian Scientific
realized within the framework of the and Technical Cooperation Program.
REFERENCES (I) MESTER, L. and MESTER, M., D-fructose : A new reducing J.Carbohydr.Nucleos.Nucleot.
i-Desoxy-l(S-hydroxy-tryptamino)sugar derivative of serotonin. 2, 141, 1975.
(2) MESTER, L., KRASKA, B., CRISBA, J. and MESTER, M., SugarAmine Interactions in the Blood Clotting System and their Effects on Haemostasis. Abstr. 5th Congr. Thrombosis and Haemostasis, Paris, 1975, p.233. (3)
FLUCKIGER, Hemoglobin
R. and WINTERHALTER, K.H., In Vitro Ale. FEBS Letters, 71, 356, 1976.
(4) KOENIG, R.J., BLOBSTEIN, S.H. and CERAMI, A., Carbohydrate of Hemoglobin Ale. J.Biol.Chem.,
Synthesis
of
Structure of 252, 2992, 1977.
(5) MESTER, L., XESTER, M., LABRID, C. and DURENG, G., Sugar Derivatives of Indolamines. Part I. : Aggregation Induced by Desoxyfructo-serotonin and its Active Transport through the Platelet Membrane. This Journal.
Purification (6) SEIDEN, S.L. and ZESTLET, J., Partial amine Oxidases from Rat Brain. Biochim.Biophys.Acta, 1962. (7) CDEBFRIEND, S., WEISSBACH, H. and of 5-Hydroxytryptamine (Serotonin) J.Biol.Chem., 215 337, 1955. -' (8) SARKAR, S., die Wirking oxydase und 1960.
of Mono2, 363,
CLARK, C.T., The Estimation in Biological Tissues.
BANERJEE, R., ISE, M.S. and ZELLER, E.A., Uber von 2-Phenylcyclopropylaminen auf die Monoaminandere Enzymsysteme. Helv.Chim.Xcta, 2, 439,
DERlVATlVES
QXlDATlON
OF INDOLAlLiINES,
OF SEROTONIN
AND ITS
PART
RAT BRAIN MONOAhilNE V.R.Gujrati
K.P.Bhargava, King
Lnstitut
Medical
George’s
d2
Chimie
(Received
Dept.of
M.Mester
and
Substances
28.10.1977; Sccepted
by
: DIFFERENCE
IN THE
DERIVATIVE
BY
OXIDASE and
College,
des
II
DESOXYFRUCTOSE
Eashee
Pharmacology,
Xii, Lucknor,
India
L .Mester :Gaturelles,
in Editor
revised W.H.
Gif-Sur-‘Yvette
form
23.2.1978.
Seegers)
Desoxyfructo-serotonin is a new synthetic sugar derivative of sero tonin, however, its formation in physiological conditions can also be expected. Using rat brain monoamine oxidase (MAO) in vitro, desoxyfructo-serotonin is significantly more slowly metabolized than serotonin. “Transcypromine”, a specific inhibitor of MAO, also inhibits the oxidative deamination of the sugar derivative.
INTRODUCTION I-Desoxy-I-(5-hydroxytryptamino)-D-fructose is a new, stable, strongly reducing sugar derivative of serotonin, which was obtained by heating serotonin oxalate with D-glucose (1) in absolute ethanol. Since 1975, the formation of similar sugar derivatives in the blood has been suggested (2). Subsequently, the existence in the blood of the desoxysugar derivative of haemoglobin has been reported (3,4), but not yet of serotonin. Nevertheless, the sugar derivative could be a possible form of circulating serotonin. Desoxyfructo-serotonin, when compared with serotonin, shor
791
SEROTOXIN
792
X-iD FiONOAHIXE
OXIDASE
Vo1.12,No.5
XETHODS Albino rats were killed by decapitation, the brain was quickly excised and homogenized in ice-cold 0.25 X sucrose with a Potter-Elvehjem homogenizer. The tissue homogenate (10X, w/v) thus obtained was centrifuged in refrigerated centrifuge at 8OOxg for 30 minutes and the supernatant decanted. The residue (A) was rehomogenized in 0.25 M sucrose and again centrifuged. The washings were mi:ed with tEe supernatant. The supernatant so obtained, was again centrifuged at 16,OOOxg for 40 minutes and supernatant decanted. The residue (B) was washed twice 0.25 M sucrose and again centrifuged at I6,ODDxg. The residue (C) soobtained consisted of mitochondrial particles and was suspended in 0.25 g sucrose (10% w/v), for the assay of MAO activity. The assay system consisted to 1.0 ml of 0.25 M phosphate buffer (pH 7.4). Suitably diluted enzyme preparations and various concentrawere incubated at 37°C tions of the substrates (5-HT or DF-5HT) The reaction was stopped by addition of I ml of 10% Eor 30 min. The tubes were centrifugated at IOOOxg for trichloroacetic acid. assayed for S-HT by the 20 min. and the supernatant was calorimetric method (7). Inhibition experiments were done using “tranylcypromine” a specific MAO inhibitor, (8) (trans-2-Phenylcyclopropylamine), The reaction mixture in a final and DF-5HT as the substrate. volume of 2 ml consisted of DF-5-HT (final concentration 0.05 z), I ml of phosphate buffer (0.5 I), suitable amount of inhibitor and 0.3 ml purified rat brain mitochondrial preparation. and water, Enzyme with the inhibitor was preincubated for 15 min. at 37°C prior to the addition of the substrate and the reaction was Activity was determined stopped after 30 min incubation. Each experiment was done in duplicate and colotimetrically (7). the mean value of the duplicate. the values represented are
RESULTS
were
The carried
rat out
brain with
AND
DISCUSSION
MAO mediated oxidation purified mitochondrial
of
5-HT and preparation
DF-5HT (6).
The substrate saturation curves of 5-HT and DF-5HT obtained from various concentrations (0.05 to 0.5 mM), were hyperbolic, thus exhibiting classical Michaelis kinetics (Fig.1). When Lineweaver-Burk curves were plotted (substrate concentrations ranging from 0.83 to 6.66 mM), the Km value of MAO for DF-5HT M) vas almost twice that of 5-HT (8.0 x low3 H) (14.28 x iO-3 This confirms that the affinity of MAO for DF-5HT is (Fig.2). considerably less when compared with 5-HT in vitro. inhibits a specific MAO inhibitor “Tranylcypromine”, (8), the oxidative deamination of DF-5HT. The results given in TABLE I, shoving 35 and 65.7 % inhibition of the MAO activity, respectively, con1irms the degradation of the sugar derivative by the enzyme MAO (Table I).
i-01.
17
,so.
5
5-HT
0.0 -
0.6-
0.4-
DF-5-HT
0.05
0
mM
0.1
0.15
SUBSTRATE
Effect of 5-HT and DF-5HT activity expressed as
0.2
0.25
0.3
0.35
0.4
CONCENTRATION
FIG. 1 concentration on rat brain change in optical density
r
>fAO
PLOT
‘LINEWEAVER -6URK
I
-- 1 = Km
5-HT h&r)
25
i 0.07
Km -14.
OF-5 28x
6’~
0~125
KIII =&oo
x 10‘~
M
.
6.0-
_A
KIII
I/ [S] Y
IO-sM
FIG. 2 Lineweaver-Burk plot showing oxidative deamination of 5-HT and DF-SHT by purified rat brain mitochondrial preparation. Initial velocity (v) was expressed as change in optical density (O.D./ 30 min.). The substrate concentration (S) was expressed as l/(S) xIO-~ M.
HT
SEROTOSIN
794
XND
ElONO~hlINE OXIDASE
I
TABLE OXIDATION Name
of
Without
*
OF
OF-5HT
inhibitor
Vo1.12,No.J
BY TRANYLCYPROMINE Concentration
INHIBITED
Z Activity
of
MAO MAO
1oo*
inhibitor
Tranylcypromine
IO -6
y.
65.0
Tranylcypromine
IO -4 M
34.3
The activity at pH 7.4 of control MAO + OF-5HT was taken as 100 z. The values in the table represent the per cent activity in presence of tranylcypromine at two different concentrations.
readily
It is conceivable metabolized and
that in vivo also DF-5HT may not be exhibit prolonged serotonin-like effects.
CONCLUSION The observed difference in the rate of oxidation of desoxyfructo-serotonin and serotonin with monoamine oxidase is due probably to a decreased affinity of the sugar derivative towards If the formation of the sugar derivative occurs in the enzyme. it may effect the metabolism and the the blood, as suggested, mode of action of serotonin in haemostasis.
ACKNOWLEDGEMENT This research was French-Indian Scientific
realized within the framework of the and Technical Cooperation Program.
REFERENCES (I) MESTER, L. and MESTER, M., D-fructose : A new reducing J.Carbohydr.Nucleos.Nucleot.
i-Desoxy-l(S-hydroxy-tryptamino)sugar derivative of serotonin. 2, 141, 1975.
(2) MESTER, L., KRASKA, B., CRISBA, J. and MESTER, M., SugarAmine Interactions in the Blood Clotting System and their Effects on Haemostasis. Abstr. 5th Congr. Thrombosis and Haemostasis, Paris, 1975, p.233. (3)
FLUCKIGER, Hemoglobin
R. and WINTERHALTER, K.H., In Vitro Ale. FEBS Letters, 71, 356, 1976.
(4) KOENIG, R.J., BLOBSTEIN, S.H. and CERAMI, A., Carbohydrate of Hemoglobin Ale. J.Biol.Chem.,
Synthesis
of
Structure of 252, 2992, 1977.
(5) MESTER, L., XESTER, M., LABRID, C. and DURENG, G., Sugar Derivatives of Indolamines. Part I. : Aggregation Induced by Desoxyfructo-serotonin and its Active Transport through the Platelet Membrane. This Journal.
Purification (6) SEIDEN, S.L. and ZESTLET, J., Partial amine Oxidases from Rat Brain. Biochim.Biophys.Acta, 1962. (7) CDEBFRIEND, S., WEISSBACH, H. and of 5-Hydroxytryptamine (Serotonin) J.Biol.Chem., 215 337, 1955. -' (8) SARKAR, S., die Wirking oxydase und 1960.
of Mono2, 363,
CLARK, C.T., The Estimation in Biological Tissues.
BANERJEE, R., ISE, M.S. and ZELLER, E.A., Uber von 2-Phenylcyclopropylaminen auf die Monoaminandere Enzymsysteme. Helv.Chim.Xcta, 2, 439,