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Observations on a histaminase of invertebrate origin: A contribution to the study of cephalopod amine oxidases
Comp. Biochem. Physiol., 1969, VoL 30, pp. 611 to 620. Pergamon Press. Printed in Great Britain
OBSERVATIONS ON A H I S T A M I N A S E OF INVERTEBRATE ORIGIN: A C O N T R I B U T I O N TO THE S T U D Y OF CEPHALOPOD A M I N E OXIDASES MARGARET C. BOADLE* Department of Pharmacology, University of Oxford, and the Marine Biological Laboratory, Plymouth
(Received 13 ffanuary 1969)
Abstract--1. The amine oxidase of the renal appendages, pancreas and optic ganglia of Eledone cirrhosa acted on histamine, ~o-N-methylhistamine and a variety of monoamines. Short-chain aliphatic diamines, e.g. putrescine and cadaverine, were not substrates. 2. The oxidation of histamine by these tissues proceeded unchanged in the presence of 10 -~ M semicarbazide. 3. The K m of the enzyme present in the renal appendages toward histamine was 6"25 x 10 -3. 4. The monocationic form of histamine was oxidized relatively more rapidly than the dicationic form by the amine oxidase of the renal appendages and pancreas.
INTRODUCTION THAT the amine oxidases take part in the biological inactivation of the amines present in mammalian tissues has been known for a long time. I n the catabolism of the catecholamines and of 5-hydroxytryptamine the enzyme known as monoamine oxidase (E.C. 1.4.3.4) plays an important part; histamine is the substrate of several mammalian enzymes of which diamine oxidase (E.C. 1.4.3.6) is one. Interest in the presence of amine oxidases in the cephalopod molluscs stems from the observations of Henze, Erspamer, von Euler and others (Henze, 1913; Erspamer, 1948, 1952; Erspamer & Boretti, 1951 ; v o n Euler, 1952; Hartman et al., 1960; Cottrell, 1967) on the occurrence in cephalopods of high concentrations of biologically active amines such as 5-hydroxytryptamine, the catecholamines, tyramine, octopamine and histamine. Some years ago Blaschko and his coworkers demonstrated the presence of an enzyme in cephalopods which attacks monoamines. T h e species studied were Eusepia ojficinalis (Blaschko, 1941; Blaschko & Himms, 1954), Loligoforbesii (Blaschko & Himms, 1954) and Octopus vulgaris (Blaschko & Hawkins, 1952). In this work a detailed investigation was made of the distribution of monoamine oxidase activity in different tissues. T h e enzymes acted on both primary and secondary monoamines; they were insensitive * Linacre College Postgraduate Student in Biochemical Pharmacology. 611
612
MARGARET C. BOADLE
to carbonyl reagents, e.g. to semicarbazide. T h e s e are properties typical of the m a m m a l i a n m o n o a m i n e oxidases and any differences f r o m the m a m m a l i a n enzymes were of the kind that can be found when oxidases f r o m different m a m m a l i a n sources are compared. I n two species of insect, Periplaneta americana L. and Blaberus discoidalis, the Malpighian tubules contain an enzyme that acts on some of the short-chain aliphatic diamines which are substrates of the m a m m a l i a n diamine oxidase (Boadle & Blaschko, 1968). T h e s e observations p r o m p t e d the present study of the amine oxidase activity found in the tissues of Eledone cirrhosa. Particular attention was given to the question whether or not aliphatic diamines or histamine were oxidized. Also, since the Malpighian tubules in insects serve an excretory function, a study of the enzymatic activity of the excretory organs of E. cirrhosa was included. S o m e of these observations have been briefly reported in an earlier note (Boadle, 1967a). M A T E R I A L S AND M E T H O D S Specimens of Eledone cirrhosa were obtained at Plymouth and from the Gatty Marine Laboratory of St. Andrews University, Fife. The material collected at Plymouth consisted of three animals the organs of which were homogenized separately and tested manometrically at Plymouth. At St. Andrews seven animals were dissected; the tissues were pooled and the frozen material was brought to Oxford where the extracts were prepared and tested. Tissues were homogenized in ice-cold 0"067 M sodium phosphate buffer of pH 7"4 and then dialysed with stirring at 4°C against the same buffer for a minimum of 4 hr. The tissue homogenates were assayed for amine oxidase activity by measuring oxygen uptake manometrically. Flasks of two different sizes were used according to the amount of tissue available. In the larger flasks the total volume of reactants was 2"0 ml; in the smaller flasks the volume was 0"8 ml. The amines tested were obtained from commercial sources as the hydrochloride or sulphate with the exception of w-N-methylhistamine hydrochloride which was very kindly prepared by Dr. Edward Gill of the Pharmacology Department, Oxford University. Enzyme activity was expressed in terms of qO2, i.e. as pl 02 consumed by 1 mg of fresh tissue/hr. Calculations were based on the initial rate of oxygen uptake usually during the first 15-20 min. The assays were all carried out at 25°C. Determinations of ammonia nitrogen were carried out by the micro-Kjeldab.1 method (Conway, 1957). The large manometer flasks were opened rapidly and 0"2 ml saturated trichloracetic acid was added to stop the reaction and absorb ammonia. The protein was precipitated at 0°C and spun down and the supernatant was then used for assay of ammonia nitrogen. RESULTS (a) Monoamines T a b l e 1 shows the results obtained w h e n a variety of tissues f r o m E. cirrhosa were tested with several m o n o a m i n e s having cyclic substituents in the side-chain. All the tissues examined showed m o n o a m i n e oxidase ( M A O ) activity, but the pancreas, liver, optic ganglia and renal appendages gave the m o s t active preparations. T h e extracts of renal appendages were also tested with three aliphatic monoamines,
613
A ttISTAMINASE OF INVERTEBRATE ORIGIN
iso-amylamine, n-amylamine and n-heptylamine; all three were oxidized giving qO2 values of 2.11, 3-84 and 4-5 respectively. TABLE I~OxIDATION
OF MONOAMINES BY TISSUE EXTRACTS FROM
E.
cirrhosa
Oxygen uptake (/zl O2/mg fresh tissue per hr) Tissue Renal appendages Pancreas Optic ganglia Liver Posterior salivary gland Stomach Caeca Heart Branchial heart Pericardial gland
Tryptamine 1"78 (2) 2"03 0-85 0"46 _+0"02 (4) 0"39 0"18 0"21 O- 1 5
---
fl-Phenylethylamine 2-49 -0"74 1"82 -----
0"56 --
N-methyl-/3phenylethylamine (2)
2"32 + 0"04 (3) 2.96 1"16 0"69 0"18 ----0"08
Number of observations given in parentheses. (b) Aliphatic diamines Members of the homologous series of aliphatic diamines, H~N [CH~] n N H 2, were tested on a number of tissue extracts. Preparations of liver and renal appendages did not act on the short-chain members of the series, putrescine (n = 4), cadaverine ( n - 5) or 1,6-diamino-n-hexane. However, with increasing chain length oxidation did take place. With the renal appendage extracts there was an oxygen uptake with 1,7-diamino-n-heptane (qO2 = 0.44) and 1,8-diamino-noctane (qO~ = 0.80). With the liver extracts a more extended series of these diamines was tested. There was no oxygen uptake with 1,7-diamino-n-heptane but the members with longer carbon chains were oxidized; the values of qOa were 0-13 for 1,8-diamino-n-octane, 0.11 for 1,9-diamino-n-nonane, 0.10 for 1,10-diamino-ndecane and 0.13 for 1,12-diamino-n-dodecane. Putrescine was tested on an extract of posterior salivary gland and of branchial heart, and cadaverine on a preparation of the optic ganglia; no oxygen uptake was observed with any of these preparations. (c) Other amines A number of amino compounds known to be substrates of a variety of amine oxidases from different sources were tested on the preparations of liver and renal appendages. T h e compounds tested with the liver preparation were L-lysine, the polyamines, spermine and spermidine, and 8-amino-n-octanoic acid; the extracts of renal appendages were tested with agmatine, spermidine, L-lysine, mescaline and 3-picolylamine (3-aminomethylpyridine). None of these amines were found to be substrates of either the liver or the renal appendage preparations. A homogenate of pancreas did not act on spermine, spermidine or agmatine.
614
MARGARET C. BOADLE
(d) Histamine and w-N-methylhistamine An investigation was made of the ability of Eledone tissues to oxidize histamine. Table 2 gives a summary of the results obtained. It can be seen that with many of the tissue extracts there was no significant uptake of oxygen in the presence of histamine. It may be mentioned that the tissues listed at the bottom of Table 2 all have monoamine oxidase activity. TABLE 2----OXIDATIONOF HISTAMINEAND~o-N-METHYLHISTAMINEBY TISSUEEXTRACTSFROM
E. cirrhosa Additional oxygen uptake (/~10~/mg fresh tissue per hr -+ S.E.) Tissue Renalappendages Pancreas Optic ganglia
Blank oxygen uptake (/~10~/mg tissue per hr -+ S.E.) 0.06_+0"01 (12) 0"04-+ 0"02 (4) 0'01 -+0"00 (4)
Histamine (10 -~ M) 0"80_+0.05(12) 1"03 _+0"05 (4) 0"14_+ 0"02 (4)
eo-N-methylhistamine (10 -2 M) 0"47-+0.01 (4) 0"81 0"10
There was no significant oxygen uptake with histamine in the presence of any of the following tissue extracts: stomach, caeca, posterior salivary gland, liver, heart, branchial heart, pericardial gland. Number of observations given in parentheses. T h r e e tissues were found in which histamine was oxidized. With the pancreas and the renal appendages there was a rapid uptake of oxygen; with the optic ganglia the rate of oxidation was less rapid. T h e mammalian enzymes responsible for the oxidation of histamine are distinguished from the classical mitochondrial monoamine oxidase by two characteristics (see Buffoni, 1967; Blaschko, 1963). Firstly, the histaminases are inhibited by carbonyl reagents such as semicarbazide whereas the classical monoamine oxidases are not inhibited by these compounds. Secondly, the histaminases act only on primary amino compounds whereas the enzymes of the second group are able to act on N-methylated amino compounds as well. W h e n the effect of semicarbazide was tested on the three tissue extracts from Eledone which had been found to possess histaminase activity, it was found that the oxidation of histamine proceeded unchanged in the presence of 10 -2 M semicarbazide. Furthermore, it was found that these same three tissues acted on the secondary amine, eo-N-methylhistamine, nearly as readily as histamine. T h e s e observations are summarized in T a b l e 2.
(e) Some properties of the histaminase activity of the renal appendages and pancreas O f the three tissues in which histarninase activity was observed enough material remained from two of them, the renal appendages and pancreas, to allow a more detailed investigation of this histaminase activity.
615
A H I S T A M I N A S E OF INVERTEBRATE O R I G I N
(i) Effect of p H on the oxidation of histamine and a monoamine by the renal appendages and pancreas. Preparations of renal appendages and pancreas were dialysed against 0.006 M sodium phosphate buffer, p H 7.4, for 1 hr and were then assayed at p H 6.0 and p H 8.0 in the presence of a 0.05 M sodium pyrophosphate buffer using as substrates histamine and a monoamine. T h e monoamine used with the renal appendages was n-heptylamine, with the pancreas, N-methyl-~-phenylethylamine. With histamine the p K of the primary amino group is 9.9 and that of the imidazole nitrogen is 6.0 (Albert, 1952). Hence, at p H 6.0 more than half of the histamine exists as a dication, whereas at p H 8.0 it is mainly in the monocationic form. On the other hand, the two monoamines tested are practically fully ionized at both values of p H used in this study. In Table 3 the ratios of the rates of oxidation of histamine at p H 6.0 and p H 8.0 have been compared. With both renal appendages and pancreas this ratio is greater at p H 6"0 than at p H 8.0 indicating an increase in the relative rate of oxidation of histamine in the presence of a higher concentration of the monocationic form of histamine at p H 8.0. TABLE 3--A COMPARISONOF THE RELATIVERATESOF OXIDATIONOF A MONOAMINEAND OF HISTAMINEBY PREPARATIONSOF RENALAPPENDAGESAND OF PANCREAS,FROME. cirrhosa AT pH 6"0 AND pH 8"0 Oxygen uptake (/~1 O2/mg fresh tissue per hr) Tissue Renal appendages Pancreas
Substrate
At pH 6"0
At pH 8"0
n-Heptylarnine Histamine n-Heptylamine : histamine N-methyl- E- phenylethylamine Histamine N-methyl-fl-phenylethylamine : histamine
1.1 0"1 11.0 2"7 0'2
5"6 1"8 3.1 9"2 3-0
13"5
3"1
With the renal appendages amine concentrations were 2 x 10 -2 M ; with the pancreas 2-5 x 10 -2 M. (ii) Competition between histamine and monoamines. T h e question arises whether histamine and the monoamines are oxidized by a single enzyme or by different enzymes. T o try to answer this question an experiment was carried out in which histamine and N-methyl-13-phenylethylamine were tested singly and together on a preparation of the renal appendages. I f the oxidation of these amines is brought about by separate enzymes, then one would expect the oxygen uptake in the presence of both amines to be the sum of that obtained with each separately. On the other hand, if one enzyme is responsible for the oxidation of both compounds, then the oxygen uptake should lie somewhere between that observed for each
616
MARGARET C . BOADLE
separately, given that zero-order conditions have been established. T h e results which are s u m m a r i z e d in T a b l e 4 show that histamine and N - m e t h y l - f l - p h e n y l ethylamine were oxidized competitively by the preparation from the renal appendages. TABLE ~
COMPETITIVE OXIDATION OF HISTAMINE AND N-METHYL-fl-PHENYLETHYLAMINE BY A PREPARATION OF THE RENAL APPENDAGES OF E. d~'rhos(2
Oxygen uptake (#10 2/mg fresh tissue per hr)
Concentration of substrate 100 mM histamine 125 m M histamine 25 m M N-methyl-fl-phenylethylamine 125 mM N-methyl-fl-phenylethylamine 100 mM histamine plus 25 m M N-methyl-flphenylethylamine 10 m M histamine 10 m M N-methyl-fl-phenylethylamine 20 mM N-methyl-fl-phenylethylamine 10 mM histamine plus 10 mM N-methyl-flphenylethylamine
1.9 1-9 6.5 7-0 6-3 0.8 3.0 4.3 3-0
!
V 4
2
|
t -i/~
o~,
o.a ,IS
FIG. 1. Lineweaver-Burk plot 1IV against 1/S for the oxidation of histamine by a dialysed preparation of renal appendages from E. cirrhosa. Abscissa: Reciprocal of the substrate concentration, S, in raM. Ordinate: Reciprocal of the velocity, V, of the enzyme reaction in/zl Os consumed/rag fresh tissue per hr. The K m of the enzyme in the renal appendages for histamine was obtained by extrapolation: -1/K,~ = -0.016.
617
A HISTAMINASE OF INVERTEBRATE ORIGIN
(iii) A~inity of the renal appendage oxidase for histamine. The K m value of the renal appendage preparation towards histamine was 6.25 x 10 -2 M (Fig. 1). (iv) Effect of inhibitors. (a) Marsilid. Inhibition of the oxidation of histamine, in a concentration of 2.5 x 10 -2 M, by a preparation of renal appendages was complete in the presence of 10 -3 M Marsilid. (b) Harmine. The oxidation of histamine, in a concentration of 2.5 x 10 -2 M, by a preparation of renal appendages was completely inhibited by 0.5 × 10 -s M harmine (see Table 5). TABLE
5--INHIBITION
BY HARMINE OF THE OXIDATION OF HISTAMINE BY A PREPARATION OF RENAL APPENDAGES FROM
E. cirrhosa
Inhibition (%) concentration of harmine Concentration of histamine 10.0 x 10-2 M 5.0 x 10-2 M 2-5 x 10 -2 M
5 x 10 -6 M
2.5 x 1 0 - 2 M
0.5 x 10 -~ M
86 91 100
71 75 80
27 39 25
(v) Ammonia production. The ammonia produced in the oxidative deamination of histamine by an extract of renal appendages was determined at the end of two experiments which were allowed to run for 60 min and 90 min and in which oxygen uptake was also measured. No precautions were carried out to prevent secondary oxidation of the aldehyde to the corresponding carboxylic acid. The ratio of the t~moles of oxygen consumed to the/zmoles of ammonia produced in these two experiments were 0-68 and 0.69 respectively (see Table 6). TABLE
6--OXYGEN
UPTAKE AND AMMONIA PRODUCTION IN THE OXIDATIVE DEAMINATION OF
HISTAMINE
(2 x
10 -2
M)
BY THE RENAL APPENDAGES OF
E. cirrhosa
Time of incubation min
/xl 02
/xmoles02
#g N2
/~molesNH3
60 97
138"5 200
5"65 8-16
115"3 163
8"3 11"7
DISCUSSION Eledone cirrhosa, like the other cephalopods which have previously been studied, contains a very active monoamine oxidase which is widely distributed in the tissues. However, the most interesting new observation made on the amine oxidase activity of this species relates to the oxidative deamination of histamine. This is brought about by the renal appendages, by the pancreas and by the optic ganglia. Histaminas¢ activity has been looked for in many invertebrate species (Boadle, 1967b), but E. cirrhosa is the first invertebrate species in which such activity has been established.
618
MARGARET C. BOADLE
Enzymes able to oxidize histamine are widely distributed in vertebrates and micro-organisms (Zeller, 1963; Buffoni, 1967). However, the histaminase activity present in the renal appendages, the pancreas and the optic ganglia of Eledone differs from the vertebrate histaminases in two important respects. In the first place the oxidation of histamine is not inhibited in the presence of 10 -3 M semicarbazide, a substance that is a strong inhibitor of the vertebrate histaminases. Secondly, the three preparations from Eledone act on ~o-N-methylhistamine, a substance that is not a substrate of vertebrate histaminases (Kapeller-Adler & Iggo, 1957; Buffoni, 1967). These observations are most readily explained by assuming that the histaminase activity is a property of a monoamine oxidase present in these tissues. In keeping with this interpretation is the observation on the renal appendage preparation in which a competitive type of oxidation of the secondary monoamine N-methyl-/3phenylethylamine and of histamine was seen. The tissue preparations from Eledone which oxidize histamine do not act on short-chain aliphatic diamines. In fact, in the homologous series of straight-chain aliphatic diamines, there was no oxidation by the renal appendages until at least seven methylene groups were present in the chain. Blaschko & Hawkins (1950) made a similar finding for rabbit liver monoamine oxidase and they suggested that the charge on the second amino group may interfere with the interaction between the enzyme and the substrate. Only as the length of the polymethylene chain separating the two amino groups becomes greater does the disturbing influence of the second amino group become less and oxidation proceed. In this connexion it is of interest that with the preparation from Eledone the relative rate of oxidation of histamine, as compared with that of a monoamine, was greater at a pH at which the histamine was predominantly in the monocationic form. The suggestion that the histaminase activity of the renal appendages of E. cirrhosa is due to the monoamine oxidase present in this tissue is also supported by the experiments in which the oxidation of histamine was studied at different values of pH. These observations lead to the conclusion that the monocationic species of histamine is the substrate of the Eledone enzyme. On the basis of similar experiments on the pig plasma histaminase (Blaschko et al., 1959), it was suggested some time ago that this enzyme acted on the monocationic form of histamine. However, this enzyme belongs to the semicarbazide-sensitive type of amine oxidases. It is known that the mammalian histaminases contain pyridoxal-5-phosphate (Blaschko & Buffoni, 1965; Buffoni, 1967). This property accounts for their inability to act on oJ-N-methylhistamine and also for their sensitivity to semicarbazide and other carbonyl reagents. In Eledone the lack of inhibition by semicarbazide as well as the fact that o~-N-methylhistamine is oxidized indicate that in this species the interaction between histamine and the enzyme does not depend on the presence of pyridoxal-5-phosphate in the active centre of the enzyme (see Blaschko & Boadle, 1968). Some time ago Bertaccini (1961) reported that in Eledone moschata treatment with several monoamine oxidase inhibitors increased the histamine content of the
A H I S T A M I N A S E OF INVERTEBRATE O R I G I N
619
optic ganglia. T h e s e observations are of interest in relation to the possible physiological significance of the histaminase activity described in E. cirrhosa. Although the affinity of the molluscan enzyme for histamine is not high, Bertaccini's findings suggest that in E. rnoschata an enzyme of the type described in E. cirrhosa is active on histamine in the living animal: it has been shown that the histaminase of the renal appendages of E. cirrhosa is inhibited by two typical inhibitors of monoamine oxidase, harmine and iproniazid (Marsilid). It is of interest that the histaminase activity of the Eledone preparations does not run parallel with their general monoamine oxidase activity. T w o possible explanations for this finding present themselves: either there are two enzymes present in the tissues, a widely distributed typical monoamine oxidase and, in addition, in the renal appendages, the pancreas and the optic ganglia, a second oxidase which is active on histamine, or alternatively the enzyme present in the three tissues with histaminase activity contains a monoamine oxidase with a substrate specificity different from that found in the other tissues investigated. T h e competition experiments described in this paper support the second interpretation, but a full answer will have to await the purification of the enzyme(s) involved. SUMMARY An amine oxidase has been found in the pancreas, renal appendages and optic ganglia of Eledone cirrhosa which is able to oxidize histamine. T h i s enzyme differs from the histaminases described in vertebrates in that it is insensitive to semicarbazide and is able to oxidize the secondary amine co-N-methylhistamine. It is suggested that the enzyme which oxidizes histamine may be identical with the monoamine oxidase also present in the pancreas, renal appendages and optic ganglia.
Acknowledgements--Thanks are due to the Director and Staff of the Marine Biological Laboratory, Plymouth, and to Dr. G. A. Cottrell of the Gatty Marine Laboratory, St. Andrews, for generous help in obtaining specimens; to Dr. Edward Gill and Mrs. Susan Penn for the preparation of the to-N-methylhistamine and to Dr. H. Blaschko for his interest and encouragement. Grants from the Oxford University Naples Biological Scholarship Fund and from the Medical Research Council are gratefully acknowledged. REFERENCES ALBERTA. (1952) Ionization, pH and biological activity. Pharmac. Rev. 4, 136-167. BERTACClNI G. (1961) Discussion in Regional 1Veurochemistry (Edited by KETY S. S. & ELKESJ.), pp. 305-306. Pergamon Press, London. BLASCHKOH. (1941) Amine oxidase in Sepia o,~cinalis, ft. Physiol., Lond. 99, 364-369. BLASCHKOH. (1963) Amine oxidase. In The Enzymes (Edited by BOnE P. D., LARDYH. & MX~BXCK K.), Vol. VIII, pp. 337-351. Academic Press, New York. BLASCHKOH. & BOADLEM. C. (1968) Substrate specificity of amine oxidases. In Pyridoxal Catalysis: Enzymes and Model Systems (Edited by SNELL E. E., BRAUNSTEINA. E., SEV~IN E. S. & TORCHINSKYYU. M.). Interscience, New York/London/Sydney. BLASCHKOH. & BUFFONI F. (1965) Pyridoxal phosphate as a constituent of the histaminase (benzylamine oxidase) of pig plasma. Proc. Roy. Soc. B 163, 45-60.
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BLASCHKOH., FaI~DM~ P. J., HAWESR. & NILSSONK. C. (1959) The amine oxidases of mammalian plasma..7. Physiol., Lond. 145, 3gA, A.94. BLASCHKOH. & HAWKINSJ. (1950) Enzymic oxidation of aliphatic diamines. Br.y. Pharmac. Chemother. 5, 625-632. BLASCHKO H. & HAWKINSJ. (1952) Observations on amine oxidase in cephalopods, y. Physiol, Lond. 118, 88-93. BLA$CHKOH. & HIMMS J. M. (1954) Enzymic oxidation of amines in decapods, y. exp. Biol. 31, 1-7. BOADLE M. C. (1967a) Observations on a histaminase of invertebrate origin, the renal appendages of Eledone cirrhosa. 3. Physiol., Lond. 192, 35-36P. BOADLEM. C. (1967b) D.Phil. Thesis, Oxford. BOADLEM. C. & BLASCHKOH. (1968) Cockroach amine oxidase: classification and substrate specificity. Comp. Biochem. Physiol. 25, 129-138. BUFFONZF. (1967) Histaminase and related amine oxidases. Pharmac. Rev. 18, 1163-1199. CONWAYE. J. (1957) Microd(~usion Analysis and Volumetric Error, 4th edn. (rev.), p. 137. Macmillan, New York. COTTRELLG. A. (1967) Occurrence of dopamine and noradrenaline in the nervous tissue of some invertebrate species. Br.oT. Pharmac. Chemother. 29, 63-69. ERSPAMEn V. (1948) Active substances in the posterior salivary glands of Octopoda--II. Tyramlne and octopamine. Acta Pharm. Tox. Kbh. 4, 224-247. ERSPAMERV. (1952) Wirksame Stoffe der hinteren Speicheldriisen der Octopoden und der Hypobranchialdriise der Purpurschnecken. Arzneimittel-Forschung 2, 253-258. ERSPAMERV. & BORETTIG. (1951) Identification and characterization by paper chromatography of enteramine, octopamine, tyramine, histamine and allied substances in extracts of posterior salivary glands of Octopoda and in other tissue extracts of vertebrates and invertebrates. Archs int. Pharmacodyn. 88, 296-332. HARTMANW. J., CLARKW. G., CYR S. D., JORDANA. L. & LEIBOLDR. A. (1960) Pharmacologically active amines and their biogenesis in the octopus. Ann. N. Y. Acad. Sci. 90, 637-666. HENZXM. (1913) p-Oxyphenl~ithylamin, das Speicheldrtisengift der Cephalopoden. HoppeSeylers Z. physiol. Chem. 87, 51-58. KAPELLER-ADLERR. & IGGOB. (1957) Histamine and its derivatives in human urine. Biochim. biophys. Acta 25, 39A, A.92. VON EULERU. S. (1952) Presence of catecholamines in visceral organs of fish and in invertebrates..4ctaphysiol, scand. 28, 297-305. ZELLERE. A. (1963) Diamlne oxidases. In The Enzymes (Edited by BoYERP. D., LARDYH. & MYRBXCKK.), Vol. VIII, pp. 313-335. Academic Press, New York.
Key Word Index--Histaminase ; monoamine oxidase; cephalopod; Eledone cirrhosa ; renal appendages; pancreas; optic ganglia.
OBSERVATIONS ON A H I S T A M I N A S E OF INVERTEBRATE ORIGIN: A C O N T R I B U T I O N TO THE S T U D Y OF CEPHALOPOD A M I N E OXIDASES MARGARET C. BOADLE* Department of Pharmacology, University of Oxford, and the Marine Biological Laboratory, Plymouth
(Received 13 ffanuary 1969)
Abstract--1. The amine oxidase of the renal appendages, pancreas and optic ganglia of Eledone cirrhosa acted on histamine, ~o-N-methylhistamine and a variety of monoamines. Short-chain aliphatic diamines, e.g. putrescine and cadaverine, were not substrates. 2. The oxidation of histamine by these tissues proceeded unchanged in the presence of 10 -~ M semicarbazide. 3. The K m of the enzyme present in the renal appendages toward histamine was 6"25 x 10 -3. 4. The monocationic form of histamine was oxidized relatively more rapidly than the dicationic form by the amine oxidase of the renal appendages and pancreas.
INTRODUCTION THAT the amine oxidases take part in the biological inactivation of the amines present in mammalian tissues has been known for a long time. I n the catabolism of the catecholamines and of 5-hydroxytryptamine the enzyme known as monoamine oxidase (E.C. 1.4.3.4) plays an important part; histamine is the substrate of several mammalian enzymes of which diamine oxidase (E.C. 1.4.3.6) is one. Interest in the presence of amine oxidases in the cephalopod molluscs stems from the observations of Henze, Erspamer, von Euler and others (Henze, 1913; Erspamer, 1948, 1952; Erspamer & Boretti, 1951 ; v o n Euler, 1952; Hartman et al., 1960; Cottrell, 1967) on the occurrence in cephalopods of high concentrations of biologically active amines such as 5-hydroxytryptamine, the catecholamines, tyramine, octopamine and histamine. Some years ago Blaschko and his coworkers demonstrated the presence of an enzyme in cephalopods which attacks monoamines. T h e species studied were Eusepia ojficinalis (Blaschko, 1941; Blaschko & Himms, 1954), Loligoforbesii (Blaschko & Himms, 1954) and Octopus vulgaris (Blaschko & Hawkins, 1952). In this work a detailed investigation was made of the distribution of monoamine oxidase activity in different tissues. T h e enzymes acted on both primary and secondary monoamines; they were insensitive * Linacre College Postgraduate Student in Biochemical Pharmacology. 611
612
MARGARET C. BOADLE
to carbonyl reagents, e.g. to semicarbazide. T h e s e are properties typical of the m a m m a l i a n m o n o a m i n e oxidases and any differences f r o m the m a m m a l i a n enzymes were of the kind that can be found when oxidases f r o m different m a m m a l i a n sources are compared. I n two species of insect, Periplaneta americana L. and Blaberus discoidalis, the Malpighian tubules contain an enzyme that acts on some of the short-chain aliphatic diamines which are substrates of the m a m m a l i a n diamine oxidase (Boadle & Blaschko, 1968). T h e s e observations p r o m p t e d the present study of the amine oxidase activity found in the tissues of Eledone cirrhosa. Particular attention was given to the question whether or not aliphatic diamines or histamine were oxidized. Also, since the Malpighian tubules in insects serve an excretory function, a study of the enzymatic activity of the excretory organs of E. cirrhosa was included. S o m e of these observations have been briefly reported in an earlier note (Boadle, 1967a). M A T E R I A L S AND M E T H O D S Specimens of Eledone cirrhosa were obtained at Plymouth and from the Gatty Marine Laboratory of St. Andrews University, Fife. The material collected at Plymouth consisted of three animals the organs of which were homogenized separately and tested manometrically at Plymouth. At St. Andrews seven animals were dissected; the tissues were pooled and the frozen material was brought to Oxford where the extracts were prepared and tested. Tissues were homogenized in ice-cold 0"067 M sodium phosphate buffer of pH 7"4 and then dialysed with stirring at 4°C against the same buffer for a minimum of 4 hr. The tissue homogenates were assayed for amine oxidase activity by measuring oxygen uptake manometrically. Flasks of two different sizes were used according to the amount of tissue available. In the larger flasks the total volume of reactants was 2"0 ml; in the smaller flasks the volume was 0"8 ml. The amines tested were obtained from commercial sources as the hydrochloride or sulphate with the exception of w-N-methylhistamine hydrochloride which was very kindly prepared by Dr. Edward Gill of the Pharmacology Department, Oxford University. Enzyme activity was expressed in terms of qO2, i.e. as pl 02 consumed by 1 mg of fresh tissue/hr. Calculations were based on the initial rate of oxygen uptake usually during the first 15-20 min. The assays were all carried out at 25°C. Determinations of ammonia nitrogen were carried out by the micro-Kjeldab.1 method (Conway, 1957). The large manometer flasks were opened rapidly and 0"2 ml saturated trichloracetic acid was added to stop the reaction and absorb ammonia. The protein was precipitated at 0°C and spun down and the supernatant was then used for assay of ammonia nitrogen. RESULTS (a) Monoamines T a b l e 1 shows the results obtained w h e n a variety of tissues f r o m E. cirrhosa were tested with several m o n o a m i n e s having cyclic substituents in the side-chain. All the tissues examined showed m o n o a m i n e oxidase ( M A O ) activity, but the pancreas, liver, optic ganglia and renal appendages gave the m o s t active preparations. T h e extracts of renal appendages were also tested with three aliphatic monoamines,
613
A ttISTAMINASE OF INVERTEBRATE ORIGIN
iso-amylamine, n-amylamine and n-heptylamine; all three were oxidized giving qO2 values of 2.11, 3-84 and 4-5 respectively. TABLE I~OxIDATION
OF MONOAMINES BY TISSUE EXTRACTS FROM
E.
cirrhosa
Oxygen uptake (/zl O2/mg fresh tissue per hr) Tissue Renal appendages Pancreas Optic ganglia Liver Posterior salivary gland Stomach Caeca Heart Branchial heart Pericardial gland
Tryptamine 1"78 (2) 2"03 0-85 0"46 _+0"02 (4) 0"39 0"18 0"21 O- 1 5
---
fl-Phenylethylamine 2-49 -0"74 1"82 -----
0"56 --
N-methyl-/3phenylethylamine (2)
2"32 + 0"04 (3) 2.96 1"16 0"69 0"18 ----0"08
Number of observations given in parentheses. (b) Aliphatic diamines Members of the homologous series of aliphatic diamines, H~N [CH~] n N H 2, were tested on a number of tissue extracts. Preparations of liver and renal appendages did not act on the short-chain members of the series, putrescine (n = 4), cadaverine ( n - 5) or 1,6-diamino-n-hexane. However, with increasing chain length oxidation did take place. With the renal appendage extracts there was an oxygen uptake with 1,7-diamino-n-heptane (qO2 = 0.44) and 1,8-diamino-noctane (qO~ = 0.80). With the liver extracts a more extended series of these diamines was tested. There was no oxygen uptake with 1,7-diamino-n-heptane but the members with longer carbon chains were oxidized; the values of qOa were 0-13 for 1,8-diamino-n-octane, 0.11 for 1,9-diamino-n-nonane, 0.10 for 1,10-diamino-ndecane and 0.13 for 1,12-diamino-n-dodecane. Putrescine was tested on an extract of posterior salivary gland and of branchial heart, and cadaverine on a preparation of the optic ganglia; no oxygen uptake was observed with any of these preparations. (c) Other amines A number of amino compounds known to be substrates of a variety of amine oxidases from different sources were tested on the preparations of liver and renal appendages. T h e compounds tested with the liver preparation were L-lysine, the polyamines, spermine and spermidine, and 8-amino-n-octanoic acid; the extracts of renal appendages were tested with agmatine, spermidine, L-lysine, mescaline and 3-picolylamine (3-aminomethylpyridine). None of these amines were found to be substrates of either the liver or the renal appendage preparations. A homogenate of pancreas did not act on spermine, spermidine or agmatine.
614
MARGARET C. BOADLE
(d) Histamine and w-N-methylhistamine An investigation was made of the ability of Eledone tissues to oxidize histamine. Table 2 gives a summary of the results obtained. It can be seen that with many of the tissue extracts there was no significant uptake of oxygen in the presence of histamine. It may be mentioned that the tissues listed at the bottom of Table 2 all have monoamine oxidase activity. TABLE 2----OXIDATIONOF HISTAMINEAND~o-N-METHYLHISTAMINEBY TISSUEEXTRACTSFROM
E. cirrhosa Additional oxygen uptake (/~10~/mg fresh tissue per hr -+ S.E.) Tissue Renalappendages Pancreas Optic ganglia
Blank oxygen uptake (/~10~/mg tissue per hr -+ S.E.) 0.06_+0"01 (12) 0"04-+ 0"02 (4) 0'01 -+0"00 (4)
Histamine (10 -~ M) 0"80_+0.05(12) 1"03 _+0"05 (4) 0"14_+ 0"02 (4)
eo-N-methylhistamine (10 -2 M) 0"47-+0.01 (4) 0"81 0"10
There was no significant oxygen uptake with histamine in the presence of any of the following tissue extracts: stomach, caeca, posterior salivary gland, liver, heart, branchial heart, pericardial gland. Number of observations given in parentheses. T h r e e tissues were found in which histamine was oxidized. With the pancreas and the renal appendages there was a rapid uptake of oxygen; with the optic ganglia the rate of oxidation was less rapid. T h e mammalian enzymes responsible for the oxidation of histamine are distinguished from the classical mitochondrial monoamine oxidase by two characteristics (see Buffoni, 1967; Blaschko, 1963). Firstly, the histaminases are inhibited by carbonyl reagents such as semicarbazide whereas the classical monoamine oxidases are not inhibited by these compounds. Secondly, the histaminases act only on primary amino compounds whereas the enzymes of the second group are able to act on N-methylated amino compounds as well. W h e n the effect of semicarbazide was tested on the three tissue extracts from Eledone which had been found to possess histaminase activity, it was found that the oxidation of histamine proceeded unchanged in the presence of 10 -2 M semicarbazide. Furthermore, it was found that these same three tissues acted on the secondary amine, eo-N-methylhistamine, nearly as readily as histamine. T h e s e observations are summarized in T a b l e 2.
(e) Some properties of the histaminase activity of the renal appendages and pancreas O f the three tissues in which histarninase activity was observed enough material remained from two of them, the renal appendages and pancreas, to allow a more detailed investigation of this histaminase activity.
615
A H I S T A M I N A S E OF INVERTEBRATE O R I G I N
(i) Effect of p H on the oxidation of histamine and a monoamine by the renal appendages and pancreas. Preparations of renal appendages and pancreas were dialysed against 0.006 M sodium phosphate buffer, p H 7.4, for 1 hr and were then assayed at p H 6.0 and p H 8.0 in the presence of a 0.05 M sodium pyrophosphate buffer using as substrates histamine and a monoamine. T h e monoamine used with the renal appendages was n-heptylamine, with the pancreas, N-methyl-~-phenylethylamine. With histamine the p K of the primary amino group is 9.9 and that of the imidazole nitrogen is 6.0 (Albert, 1952). Hence, at p H 6.0 more than half of the histamine exists as a dication, whereas at p H 8.0 it is mainly in the monocationic form. On the other hand, the two monoamines tested are practically fully ionized at both values of p H used in this study. In Table 3 the ratios of the rates of oxidation of histamine at p H 6.0 and p H 8.0 have been compared. With both renal appendages and pancreas this ratio is greater at p H 6"0 than at p H 8.0 indicating an increase in the relative rate of oxidation of histamine in the presence of a higher concentration of the monocationic form of histamine at p H 8.0. TABLE 3--A COMPARISONOF THE RELATIVERATESOF OXIDATIONOF A MONOAMINEAND OF HISTAMINEBY PREPARATIONSOF RENALAPPENDAGESAND OF PANCREAS,FROME. cirrhosa AT pH 6"0 AND pH 8"0 Oxygen uptake (/~1 O2/mg fresh tissue per hr) Tissue Renal appendages Pancreas
Substrate
At pH 6"0
At pH 8"0
n-Heptylarnine Histamine n-Heptylamine : histamine N-methyl- E- phenylethylamine Histamine N-methyl-fl-phenylethylamine : histamine
1.1 0"1 11.0 2"7 0'2
5"6 1"8 3.1 9"2 3-0
13"5
3"1
With the renal appendages amine concentrations were 2 x 10 -2 M ; with the pancreas 2-5 x 10 -2 M. (ii) Competition between histamine and monoamines. T h e question arises whether histamine and the monoamines are oxidized by a single enzyme or by different enzymes. T o try to answer this question an experiment was carried out in which histamine and N-methyl-13-phenylethylamine were tested singly and together on a preparation of the renal appendages. I f the oxidation of these amines is brought about by separate enzymes, then one would expect the oxygen uptake in the presence of both amines to be the sum of that obtained with each separately. On the other hand, if one enzyme is responsible for the oxidation of both compounds, then the oxygen uptake should lie somewhere between that observed for each
616
MARGARET C . BOADLE
separately, given that zero-order conditions have been established. T h e results which are s u m m a r i z e d in T a b l e 4 show that histamine and N - m e t h y l - f l - p h e n y l ethylamine were oxidized competitively by the preparation from the renal appendages. TABLE ~
COMPETITIVE OXIDATION OF HISTAMINE AND N-METHYL-fl-PHENYLETHYLAMINE BY A PREPARATION OF THE RENAL APPENDAGES OF E. d~'rhos(2
Oxygen uptake (#10 2/mg fresh tissue per hr)
Concentration of substrate 100 mM histamine 125 m M histamine 25 m M N-methyl-fl-phenylethylamine 125 mM N-methyl-fl-phenylethylamine 100 mM histamine plus 25 m M N-methyl-flphenylethylamine 10 m M histamine 10 m M N-methyl-fl-phenylethylamine 20 mM N-methyl-fl-phenylethylamine 10 mM histamine plus 10 mM N-methyl-flphenylethylamine
1.9 1-9 6.5 7-0 6-3 0.8 3.0 4.3 3-0
!
V 4
2
|
t -i/~
o~,
o.a ,IS
FIG. 1. Lineweaver-Burk plot 1IV against 1/S for the oxidation of histamine by a dialysed preparation of renal appendages from E. cirrhosa. Abscissa: Reciprocal of the substrate concentration, S, in raM. Ordinate: Reciprocal of the velocity, V, of the enzyme reaction in/zl Os consumed/rag fresh tissue per hr. The K m of the enzyme in the renal appendages for histamine was obtained by extrapolation: -1/K,~ = -0.016.
617
A HISTAMINASE OF INVERTEBRATE ORIGIN
(iii) A~inity of the renal appendage oxidase for histamine. The K m value of the renal appendage preparation towards histamine was 6.25 x 10 -2 M (Fig. 1). (iv) Effect of inhibitors. (a) Marsilid. Inhibition of the oxidation of histamine, in a concentration of 2.5 x 10 -2 M, by a preparation of renal appendages was complete in the presence of 10 -3 M Marsilid. (b) Harmine. The oxidation of histamine, in a concentration of 2.5 x 10 -2 M, by a preparation of renal appendages was completely inhibited by 0.5 × 10 -s M harmine (see Table 5). TABLE
5--INHIBITION
BY HARMINE OF THE OXIDATION OF HISTAMINE BY A PREPARATION OF RENAL APPENDAGES FROM
E. cirrhosa
Inhibition (%) concentration of harmine Concentration of histamine 10.0 x 10-2 M 5.0 x 10-2 M 2-5 x 10 -2 M
5 x 10 -6 M
2.5 x 1 0 - 2 M
0.5 x 10 -~ M
86 91 100
71 75 80
27 39 25
(v) Ammonia production. The ammonia produced in the oxidative deamination of histamine by an extract of renal appendages was determined at the end of two experiments which were allowed to run for 60 min and 90 min and in which oxygen uptake was also measured. No precautions were carried out to prevent secondary oxidation of the aldehyde to the corresponding carboxylic acid. The ratio of the t~moles of oxygen consumed to the/zmoles of ammonia produced in these two experiments were 0-68 and 0.69 respectively (see Table 6). TABLE
6--OXYGEN
UPTAKE AND AMMONIA PRODUCTION IN THE OXIDATIVE DEAMINATION OF
HISTAMINE
(2 x
10 -2
M)
BY THE RENAL APPENDAGES OF
E. cirrhosa
Time of incubation min
/xl 02
/xmoles02
#g N2
/~molesNH3
60 97
138"5 200
5"65 8-16
115"3 163
8"3 11"7
DISCUSSION Eledone cirrhosa, like the other cephalopods which have previously been studied, contains a very active monoamine oxidase which is widely distributed in the tissues. However, the most interesting new observation made on the amine oxidase activity of this species relates to the oxidative deamination of histamine. This is brought about by the renal appendages, by the pancreas and by the optic ganglia. Histaminas¢ activity has been looked for in many invertebrate species (Boadle, 1967b), but E. cirrhosa is the first invertebrate species in which such activity has been established.
618
MARGARET C. BOADLE
Enzymes able to oxidize histamine are widely distributed in vertebrates and micro-organisms (Zeller, 1963; Buffoni, 1967). However, the histaminase activity present in the renal appendages, the pancreas and the optic ganglia of Eledone differs from the vertebrate histaminases in two important respects. In the first place the oxidation of histamine is not inhibited in the presence of 10 -3 M semicarbazide, a substance that is a strong inhibitor of the vertebrate histaminases. Secondly, the three preparations from Eledone act on ~o-N-methylhistamine, a substance that is not a substrate of vertebrate histaminases (Kapeller-Adler & Iggo, 1957; Buffoni, 1967). These observations are most readily explained by assuming that the histaminase activity is a property of a monoamine oxidase present in these tissues. In keeping with this interpretation is the observation on the renal appendage preparation in which a competitive type of oxidation of the secondary monoamine N-methyl-/3phenylethylamine and of histamine was seen. The tissue preparations from Eledone which oxidize histamine do not act on short-chain aliphatic diamines. In fact, in the homologous series of straight-chain aliphatic diamines, there was no oxidation by the renal appendages until at least seven methylene groups were present in the chain. Blaschko & Hawkins (1950) made a similar finding for rabbit liver monoamine oxidase and they suggested that the charge on the second amino group may interfere with the interaction between the enzyme and the substrate. Only as the length of the polymethylene chain separating the two amino groups becomes greater does the disturbing influence of the second amino group become less and oxidation proceed. In this connexion it is of interest that with the preparation from Eledone the relative rate of oxidation of histamine, as compared with that of a monoamine, was greater at a pH at which the histamine was predominantly in the monocationic form. The suggestion that the histaminase activity of the renal appendages of E. cirrhosa is due to the monoamine oxidase present in this tissue is also supported by the experiments in which the oxidation of histamine was studied at different values of pH. These observations lead to the conclusion that the monocationic species of histamine is the substrate of the Eledone enzyme. On the basis of similar experiments on the pig plasma histaminase (Blaschko et al., 1959), it was suggested some time ago that this enzyme acted on the monocationic form of histamine. However, this enzyme belongs to the semicarbazide-sensitive type of amine oxidases. It is known that the mammalian histaminases contain pyridoxal-5-phosphate (Blaschko & Buffoni, 1965; Buffoni, 1967). This property accounts for their inability to act on oJ-N-methylhistamine and also for their sensitivity to semicarbazide and other carbonyl reagents. In Eledone the lack of inhibition by semicarbazide as well as the fact that o~-N-methylhistamine is oxidized indicate that in this species the interaction between histamine and the enzyme does not depend on the presence of pyridoxal-5-phosphate in the active centre of the enzyme (see Blaschko & Boadle, 1968). Some time ago Bertaccini (1961) reported that in Eledone moschata treatment with several monoamine oxidase inhibitors increased the histamine content of the
A H I S T A M I N A S E OF INVERTEBRATE O R I G I N
619
optic ganglia. T h e s e observations are of interest in relation to the possible physiological significance of the histaminase activity described in E. cirrhosa. Although the affinity of the molluscan enzyme for histamine is not high, Bertaccini's findings suggest that in E. rnoschata an enzyme of the type described in E. cirrhosa is active on histamine in the living animal: it has been shown that the histaminase of the renal appendages of E. cirrhosa is inhibited by two typical inhibitors of monoamine oxidase, harmine and iproniazid (Marsilid). It is of interest that the histaminase activity of the Eledone preparations does not run parallel with their general monoamine oxidase activity. T w o possible explanations for this finding present themselves: either there are two enzymes present in the tissues, a widely distributed typical monoamine oxidase and, in addition, in the renal appendages, the pancreas and the optic ganglia, a second oxidase which is active on histamine, or alternatively the enzyme present in the three tissues with histaminase activity contains a monoamine oxidase with a substrate specificity different from that found in the other tissues investigated. T h e competition experiments described in this paper support the second interpretation, but a full answer will have to await the purification of the enzyme(s) involved. SUMMARY An amine oxidase has been found in the pancreas, renal appendages and optic ganglia of Eledone cirrhosa which is able to oxidize histamine. T h i s enzyme differs from the histaminases described in vertebrates in that it is insensitive to semicarbazide and is able to oxidize the secondary amine co-N-methylhistamine. It is suggested that the enzyme which oxidizes histamine may be identical with the monoamine oxidase also present in the pancreas, renal appendages and optic ganglia.
Acknowledgements--Thanks are due to the Director and Staff of the Marine Biological Laboratory, Plymouth, and to Dr. G. A. Cottrell of the Gatty Marine Laboratory, St. Andrews, for generous help in obtaining specimens; to Dr. Edward Gill and Mrs. Susan Penn for the preparation of the to-N-methylhistamine and to Dr. H. Blaschko for his interest and encouragement. Grants from the Oxford University Naples Biological Scholarship Fund and from the Medical Research Council are gratefully acknowledged. REFERENCES ALBERTA. (1952) Ionization, pH and biological activity. Pharmac. Rev. 4, 136-167. BERTACClNI G. (1961) Discussion in Regional 1Veurochemistry (Edited by KETY S. S. & ELKESJ.), pp. 305-306. Pergamon Press, London. BLASCHKOH. (1941) Amine oxidase in Sepia o,~cinalis, ft. Physiol., Lond. 99, 364-369. BLASCHKOH. (1963) Amine oxidase. In The Enzymes (Edited by BOnE P. D., LARDYH. & MX~BXCK K.), Vol. VIII, pp. 337-351. Academic Press, New York. BLASCHKOH. & BOADLEM. C. (1968) Substrate specificity of amine oxidases. In Pyridoxal Catalysis: Enzymes and Model Systems (Edited by SNELL E. E., BRAUNSTEINA. E., SEV~IN E. S. & TORCHINSKYYU. M.). Interscience, New York/London/Sydney. BLASCHKOH. & BUFFONI F. (1965) Pyridoxal phosphate as a constituent of the histaminase (benzylamine oxidase) of pig plasma. Proc. Roy. Soc. B 163, 45-60.
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MARGARETC. BOADLE
BLASCHKOH., FaI~DM~ P. J., HAWESR. & NILSSONK. C. (1959) The amine oxidases of mammalian plasma..7. Physiol., Lond. 145, 3gA, A.94. BLASCHKOH. & HAWKINSJ. (1950) Enzymic oxidation of aliphatic diamines. Br.y. Pharmac. Chemother. 5, 625-632. BLASCHKO H. & HAWKINSJ. (1952) Observations on amine oxidase in cephalopods, y. Physiol, Lond. 118, 88-93. BLA$CHKOH. & HIMMS J. M. (1954) Enzymic oxidation of amines in decapods, y. exp. Biol. 31, 1-7. BOADLE M. C. (1967a) Observations on a histaminase of invertebrate origin, the renal appendages of Eledone cirrhosa. 3. Physiol., Lond. 192, 35-36P. BOADLEM. C. (1967b) D.Phil. Thesis, Oxford. BOADLEM. C. & BLASCHKOH. (1968) Cockroach amine oxidase: classification and substrate specificity. Comp. Biochem. Physiol. 25, 129-138. BUFFONZF. (1967) Histaminase and related amine oxidases. Pharmac. Rev. 18, 1163-1199. CONWAYE. J. (1957) Microd(~usion Analysis and Volumetric Error, 4th edn. (rev.), p. 137. Macmillan, New York. COTTRELLG. A. (1967) Occurrence of dopamine and noradrenaline in the nervous tissue of some invertebrate species. Br.oT. Pharmac. Chemother. 29, 63-69. ERSPAMEn V. (1948) Active substances in the posterior salivary glands of Octopoda--II. Tyramlne and octopamine. Acta Pharm. Tox. Kbh. 4, 224-247. ERSPAMERV. (1952) Wirksame Stoffe der hinteren Speicheldriisen der Octopoden und der Hypobranchialdriise der Purpurschnecken. Arzneimittel-Forschung 2, 253-258. ERSPAMERV. & BORETTIG. (1951) Identification and characterization by paper chromatography of enteramine, octopamine, tyramine, histamine and allied substances in extracts of posterior salivary glands of Octopoda and in other tissue extracts of vertebrates and invertebrates. Archs int. Pharmacodyn. 88, 296-332. HARTMANW. J., CLARKW. G., CYR S. D., JORDANA. L. & LEIBOLDR. A. (1960) Pharmacologically active amines and their biogenesis in the octopus. Ann. N. Y. Acad. Sci. 90, 637-666. HENZXM. (1913) p-Oxyphenl~ithylamin, das Speicheldrtisengift der Cephalopoden. HoppeSeylers Z. physiol. Chem. 87, 51-58. KAPELLER-ADLERR. & IGGOB. (1957) Histamine and its derivatives in human urine. Biochim. biophys. Acta 25, 39A, A.92. VON EULERU. S. (1952) Presence of catecholamines in visceral organs of fish and in invertebrates..4ctaphysiol, scand. 28, 297-305. ZELLERE. A. (1963) Diamlne oxidases. In The Enzymes (Edited by BoYERP. D., LARDYH. & MYRBXCKK.), Vol. VIII, pp. 313-335. Academic Press, New York.
Key Word Index--Histaminase ; monoamine oxidase; cephalopod; Eledone cirrhosa ; renal appendages; pancreas; optic ganglia.