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Precursors of histidine dipeptides in molluscan tissues
Comp. Biochem. Physiol., 1972, Vol. 41B, pp. 453 to 456. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION PRECURSORS OF H I S T I D I N E D I P E P T I D E S IN MOLLUSCAN TISSUES A. A. BOLDYREV and A. V. LEBEDEV Department of Animal Biochemistry of the Moscow State University, Moscow W-234, U.S.S.R. (Received 28 July 1971) Abstract--1. There are no dipeptides (carnosine and anserine) among extractive substances of the muscles of some molluscan invertebrates. 2. The precursors of dipeptides--fl-alanine and histidine---are present in tissues of Mollusca that do not contain y-NH2-butyric acid. 3. Muscles of Helixpomatia together only have fl-alanine, histidine, 1-methyl histidine and 3-methyl histidine. 4. More often these compounds are discovered separately. Their content in some cases is as much as 150-300 mg %. INTRODUCTION THE BIOLOGICALrole of histidine dipeptides---carnosine and anserine--is not yet established fully. These compounds are only found (in quantity) in skeletal muscles of vertebrates (Boldyrev & Severin, 1955; Severin, 1957; Crush, 1970). Their content depends on the type and functional role of the muscles; the formation of dipeptides from the precursors is in close agreement in time with a reflex arc circuit and also with the beginning of the functioning of ion pumps in muscles (Severin & Fedorova, 1952; Vulphson, 1952; Severin, 1967; Fanburg et al., 1958). Investigations of the occurrence of histidine dipeptides and some of their amino acid precursors in vertebrate muscles has led to the conclusion about the "effect of functional substitution": some species have a lot of anserine and only traces of carnosine whilst other species have a high content of histidine (or fl-alanine) and no dipeptides (Vulphson, 1952; Crush, 1970). Moreover, the functional and structural complication of the myoneural junction of vertebrates is accompanied by the substitution of more simple precursors--histidine and fl-alanine by their derivatives carnosine and anserine (Boldyrev & Sever[n, 1955; Crush, 1970). Analysis of the influence of histidine compounds on various reflexions of muscle function suggests a relationship between the appearance of these compounds and the formation of cholinergic membranes (in the evolution process). In this connexion we studied the content of histidine dipeptides and their precursors in invertebrate tissues which possess both cholinergic and noncholinergic types of excitation. Such an investigation may show the time of the phylogenetic appearance of these compounds and give further confirmation of the 453
454
A. A. BOLDYREV AND A. V. LEBEDEV
interconnexion between histidine compounds and the specific type of exciting membranes. We found carnosine and anserine to be absent among the nitrogenous extractive substances of muscles of invertebrates under study. Their precursors, /3alanine and histidine (and 1-methyl histidine, 3-methyl histidine sometimes), are present in notable amounts in Mollusca only. In this paper evidence about the precursors of dipeptides in Mollusca tissues is presented and discussed. MATERIALS AND METHODS The results of tissue analysis of ten species of molluscs (from three classes) are given here. For a more complete review on nitrogenous extractive substances in invertebrate muscles see Lebedev (1971). All species were collected in the middle of August 1968 in Peter the Great Gulf (the Far East of U.S.S.R.) with the exceptions of Helix and Anodonta, which were obtained from Supply of Biological Materials (Moscow). One of the specimens of Ommastrepheswas bought in a food shop. All of the samples were treated according to Wood (1958) and analysed in an automatic amino analyser "ILC-3BC, Jeol" (Japan) (Moore, et al,. 1958); the limit of sensitivity for ninhydrin-positive substances being 10 .8 mole/1. RESULTS AND DISCUSSION The results of our analysis are presented in Table 1. There was no carnosine and/or anserine present in the muscles of the specimens investigated. This was shown earlier by us on a wider range of invertebrates (Lebedev & Boldyrev, 1971). The dipeptide precursors were discovered in a great variety, but only in small amounts, in the podium muscles of Helix. Most commonly, histidine (Modiolus adductor, Neptunea podium, Ommastrephes mantle) or /3-alanine (striated adductor of Patinopecten, adductor of Ostrea, muscles of Octopus) is discovered in the muscle tissue of Mollusca separately. Muscles of all species (apart from Bivalvia) do not contain 7,-NH~-butyric acid. This is in agreement with the absence of the "glutamic type" transmission of excitation, which is characteristic of most of the Mollusca. We have shown on a great variety of invertebrates that v-NH2-butyric acid is never present together with histidine or fl-alanine (Lebedev & Boldyrev, 1971). In this connexion we investigated the cerebral ganglion of Octopus, which contains a large amount of acetylcholine (Loe & Florey, 1966): ,,.2/xmole/g of tissue. This sample had more than I00 mg % of fl-alanine, a little histidine and no 7-NH~-butyric acid (see Table 1). This confirms that ),-NH~-butyric acid is coupled with noncholinergic structures, but that the dipeptide precursors are coupled with cholinergic structures. It is noteworthy that some Mollusca contain just as much histidine (Ommustrephes) or/3-alanine (Ostrea, Octopus) as some Piscea (Vulphson, 1962). At the same time the content of the essential amino acids (for example phenyl-ala) is uniform in different specimens (see Table 1). Thus the pattern of amino acid precursors of dipeptides in invertebrates is the same as the pattern of dipeptides in vertebrates. It is possible to suppose that the
Adductor muscle Adductor muscle Adductor muscle Podium muscle Podium muscle Podium muscle Podium muscle 10.8 2.1 151.0 4.7
4.7
2.7
4.5
0.8
fl-ala
* Specimen was bought in a food shop (fresh-freezing).
Smooth(helical) Smooth(helical) Smooth(helical) Cerebral ganglion Smooth(helical) Smooth (helical) Smooth (helical) Smooth(helical) Smooth(helical)
Smooth paramyos. 343.5 287.0 265"0 101-0 5-7 2"6 5.4 4.2
Traces
Smooth paramyos. (white) Absent Smooth paramyos. (pigmented) 2"4
Striated Smooth paramyos. Striated + smooth Smooth paramyos.
Smooth paramyos., more tonic Smooth paramyos., less tonic
Adductor muscle
Adductor muscle
Smooth paramyos. Smooth paramyos. Smooth paramyos.
Type
Adductor muscle Podium muscle Adductor muscle
Tissue
1.9 2-4 2.8 3.4 5.6 9"4 64.0 71-0 62.0
3.2
43.5 21"0
5.2 1.8 6"9 Absent
20.7
11.2
0.9 0.7 5-2
his
0-6
Absent 6.8
Absent Absent Absent Absent
Absent
Absent
Absent Absent Absent
3-CH 3 his
Absent Absent Absent Absent Absent Absent Absent Absent Absent
Absent
Absent Absent
Absent Absent Absent Absent
Traces
Absent
0.8 1"8 6.4
3.5 2.0 2.7 5.4 7.8 5"4 5.4 5.4
1.7
11.1 10-9
5.5 3.9 3.7 2-6
3-3
3.6
4.0
1-1
23.4
25"2
25.8
20.8 21.0
21-3
18.3
15"5 19.4 19-6
~,-NH 2 Phenylbut ala Dry wt.
(mg/100 g OF TISSUE)
Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent
8.4
Absent Absent
Absent Absent Absent Absent
Absent
Absent
Traces 2"4 Absent
1-CH 3 his
OF SOME EXTRACTIVE NITROGENOUS COMPOUNDS I N MOLLUSCAN TISSUES
Tentacle muscle (lst) Tentacle muscle (2nd) Mantle muscle (2nd) Nerve tissue (2rid) Ommastrephes (Sloanei) Tentacle muscle (lst) pacificus Tentacle muscle (2nd) Mantle muscle (lst) Mantle muscle (2nd) Mantle muscle (3rd)*
Helix pomatia Octopus conispadicus
Ostrea gigas Acmea sp. Neptunea sp.
Patinopecten yessoensis
mytilua grayanus modiolus modiolus
Anodonta cygnea
Species
TABLE I--CONTENT
q~
.
o~ rr-
a~
o
o
456
A. A. BOLDYREVAND A. V. LEBEDEV
properties of this pattern are connected rather with the specific (cholinergic) type of excitation than with the level of development of the contractile apparatus. I n this context the appearance of carnosine and subsequently anserine (in Chordata) seems to be connected with a complication of the cholinergic structures. REFERENCES BOLDY~V A. A. & SEVERINS. E. (1966) Imidazole compounds of muscles and metabolic pathway in skeletal muscle. Biologicheskie nauki (USSR). 4, 54-74. CRUSH K. G. (1970) Carnosine and related substances in animal tissues. Comp. Biochem. Physiol. 34, 3-30. FANBURG B. L., D ~ C H M ~ D. B., MOLL D. & ROTH S. I. (1968) Calcium transport in isolated sarcoplasmic reticulum during muscle maturation. Nature, Lond. 218, 962-964. LEBErmV A. V. (1971) Nitrogenous extractive compounds of muscle tissue of invertebrates. Jurnal evoljuzionnoj Biokhimii i Physiologii (USSR). (In press.) LEBEDEV A. V. & BOLDY~V A. A. (1972) Precursors of histidine containing dipeptides in muscle tissue of invertebrates. Biokhimija (USSR). 37, 1. LoE P.-R. & FLOm~YE. (1966) The distribution of acetylcholine and cholinesterase in the nervous system and in innervated organs of Octopus dofleini. Comp. Biochem. Physiol. 17, 509-522. MOORE S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. Analyt. Chem. 30, 1185-1190. SEVERIN S. E. (1967) The participation of natural imidazole containing dipeptides in biosynthesis and reception of acetylcholine. Uspekhi sovremennoj biologii (USSR). 64, 181-196. SEVERIN S. E. & FEDOROVAV. N. (1952) Content of carnosine, anserine, histidine and fl-alanine in skeletal muscle of hen in embrionic development. Dokl. Acad. Nauk Sci. S S S R 82, 443-446. VULPHSONP. L. (1962) Comparative biochemical analysis of amino acids and dipeptides in muscle tissue. Uspekhi biologieheskoj khimii (USSR). 4, 82-92. WOOD J. D. (1958) Nitrogen excretion in some marine teleosts. Can. J. Biochem. Physiol. 36, 1237-1242.
Key Word Index--Histidine dipeptides; carnosine; anserine; cholinergic; Helix pomatia ; Anodonta cygnea ; molluscs; Ommastrephes pacificus.
SHORT COMMUNICATION PRECURSORS OF H I S T I D I N E D I P E P T I D E S IN MOLLUSCAN TISSUES A. A. BOLDYREV and A. V. LEBEDEV Department of Animal Biochemistry of the Moscow State University, Moscow W-234, U.S.S.R. (Received 28 July 1971) Abstract--1. There are no dipeptides (carnosine and anserine) among extractive substances of the muscles of some molluscan invertebrates. 2. The precursors of dipeptides--fl-alanine and histidine---are present in tissues of Mollusca that do not contain y-NH2-butyric acid. 3. Muscles of Helixpomatia together only have fl-alanine, histidine, 1-methyl histidine and 3-methyl histidine. 4. More often these compounds are discovered separately. Their content in some cases is as much as 150-300 mg %. INTRODUCTION THE BIOLOGICALrole of histidine dipeptides---carnosine and anserine--is not yet established fully. These compounds are only found (in quantity) in skeletal muscles of vertebrates (Boldyrev & Severin, 1955; Severin, 1957; Crush, 1970). Their content depends on the type and functional role of the muscles; the formation of dipeptides from the precursors is in close agreement in time with a reflex arc circuit and also with the beginning of the functioning of ion pumps in muscles (Severin & Fedorova, 1952; Vulphson, 1952; Severin, 1967; Fanburg et al., 1958). Investigations of the occurrence of histidine dipeptides and some of their amino acid precursors in vertebrate muscles has led to the conclusion about the "effect of functional substitution": some species have a lot of anserine and only traces of carnosine whilst other species have a high content of histidine (or fl-alanine) and no dipeptides (Vulphson, 1952; Crush, 1970). Moreover, the functional and structural complication of the myoneural junction of vertebrates is accompanied by the substitution of more simple precursors--histidine and fl-alanine by their derivatives carnosine and anserine (Boldyrev & Sever[n, 1955; Crush, 1970). Analysis of the influence of histidine compounds on various reflexions of muscle function suggests a relationship between the appearance of these compounds and the formation of cholinergic membranes (in the evolution process). In this connexion we studied the content of histidine dipeptides and their precursors in invertebrate tissues which possess both cholinergic and noncholinergic types of excitation. Such an investigation may show the time of the phylogenetic appearance of these compounds and give further confirmation of the 453
454
A. A. BOLDYREV AND A. V. LEBEDEV
interconnexion between histidine compounds and the specific type of exciting membranes. We found carnosine and anserine to be absent among the nitrogenous extractive substances of muscles of invertebrates under study. Their precursors, /3alanine and histidine (and 1-methyl histidine, 3-methyl histidine sometimes), are present in notable amounts in Mollusca only. In this paper evidence about the precursors of dipeptides in Mollusca tissues is presented and discussed. MATERIALS AND METHODS The results of tissue analysis of ten species of molluscs (from three classes) are given here. For a more complete review on nitrogenous extractive substances in invertebrate muscles see Lebedev (1971). All species were collected in the middle of August 1968 in Peter the Great Gulf (the Far East of U.S.S.R.) with the exceptions of Helix and Anodonta, which were obtained from Supply of Biological Materials (Moscow). One of the specimens of Ommastrepheswas bought in a food shop. All of the samples were treated according to Wood (1958) and analysed in an automatic amino analyser "ILC-3BC, Jeol" (Japan) (Moore, et al,. 1958); the limit of sensitivity for ninhydrin-positive substances being 10 .8 mole/1. RESULTS AND DISCUSSION The results of our analysis are presented in Table 1. There was no carnosine and/or anserine present in the muscles of the specimens investigated. This was shown earlier by us on a wider range of invertebrates (Lebedev & Boldyrev, 1971). The dipeptide precursors were discovered in a great variety, but only in small amounts, in the podium muscles of Helix. Most commonly, histidine (Modiolus adductor, Neptunea podium, Ommastrephes mantle) or /3-alanine (striated adductor of Patinopecten, adductor of Ostrea, muscles of Octopus) is discovered in the muscle tissue of Mollusca separately. Muscles of all species (apart from Bivalvia) do not contain 7,-NH~-butyric acid. This is in agreement with the absence of the "glutamic type" transmission of excitation, which is characteristic of most of the Mollusca. We have shown on a great variety of invertebrates that v-NH2-butyric acid is never present together with histidine or fl-alanine (Lebedev & Boldyrev, 1971). In this connexion we investigated the cerebral ganglion of Octopus, which contains a large amount of acetylcholine (Loe & Florey, 1966): ,,.2/xmole/g of tissue. This sample had more than I00 mg % of fl-alanine, a little histidine and no 7-NH~-butyric acid (see Table 1). This confirms that ),-NH~-butyric acid is coupled with noncholinergic structures, but that the dipeptide precursors are coupled with cholinergic structures. It is noteworthy that some Mollusca contain just as much histidine (Ommustrephes) or/3-alanine (Ostrea, Octopus) as some Piscea (Vulphson, 1962). At the same time the content of the essential amino acids (for example phenyl-ala) is uniform in different specimens (see Table 1). Thus the pattern of amino acid precursors of dipeptides in invertebrates is the same as the pattern of dipeptides in vertebrates. It is possible to suppose that the
Adductor muscle Adductor muscle Adductor muscle Podium muscle Podium muscle Podium muscle Podium muscle 10.8 2.1 151.0 4.7
4.7
2.7
4.5
0.8
fl-ala
* Specimen was bought in a food shop (fresh-freezing).
Smooth(helical) Smooth(helical) Smooth(helical) Cerebral ganglion Smooth(helical) Smooth (helical) Smooth (helical) Smooth(helical) Smooth(helical)
Smooth paramyos. 343.5 287.0 265"0 101-0 5-7 2"6 5.4 4.2
Traces
Smooth paramyos. (white) Absent Smooth paramyos. (pigmented) 2"4
Striated Smooth paramyos. Striated + smooth Smooth paramyos.
Smooth paramyos., more tonic Smooth paramyos., less tonic
Adductor muscle
Adductor muscle
Smooth paramyos. Smooth paramyos. Smooth paramyos.
Type
Adductor muscle Podium muscle Adductor muscle
Tissue
1.9 2-4 2.8 3.4 5.6 9"4 64.0 71-0 62.0
3.2
43.5 21"0
5.2 1.8 6"9 Absent
20.7
11.2
0.9 0.7 5-2
his
0-6
Absent 6.8
Absent Absent Absent Absent
Absent
Absent
Absent Absent Absent
3-CH 3 his
Absent Absent Absent Absent Absent Absent Absent Absent Absent
Absent
Absent Absent
Absent Absent Absent Absent
Traces
Absent
0.8 1"8 6.4
3.5 2.0 2.7 5.4 7.8 5"4 5.4 5.4
1.7
11.1 10-9
5.5 3.9 3.7 2-6
3-3
3.6
4.0
1-1
23.4
25"2
25.8
20.8 21.0
21-3
18.3
15"5 19.4 19-6
~,-NH 2 Phenylbut ala Dry wt.
(mg/100 g OF TISSUE)
Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent
8.4
Absent Absent
Absent Absent Absent Absent
Absent
Absent
Traces 2"4 Absent
1-CH 3 his
OF SOME EXTRACTIVE NITROGENOUS COMPOUNDS I N MOLLUSCAN TISSUES
Tentacle muscle (lst) Tentacle muscle (2nd) Mantle muscle (2nd) Nerve tissue (2rid) Ommastrephes (Sloanei) Tentacle muscle (lst) pacificus Tentacle muscle (2nd) Mantle muscle (lst) Mantle muscle (2nd) Mantle muscle (3rd)*
Helix pomatia Octopus conispadicus
Ostrea gigas Acmea sp. Neptunea sp.
Patinopecten yessoensis
mytilua grayanus modiolus modiolus
Anodonta cygnea
Species
TABLE I--CONTENT
q~
.
o~ rr-
a~
o
o
456
A. A. BOLDYREVAND A. V. LEBEDEV
properties of this pattern are connected rather with the specific (cholinergic) type of excitation than with the level of development of the contractile apparatus. I n this context the appearance of carnosine and subsequently anserine (in Chordata) seems to be connected with a complication of the cholinergic structures. REFERENCES BOLDY~V A. A. & SEVERINS. E. (1966) Imidazole compounds of muscles and metabolic pathway in skeletal muscle. Biologicheskie nauki (USSR). 4, 54-74. CRUSH K. G. (1970) Carnosine and related substances in animal tissues. Comp. Biochem. Physiol. 34, 3-30. FANBURG B. L., D ~ C H M ~ D. B., MOLL D. & ROTH S. I. (1968) Calcium transport in isolated sarcoplasmic reticulum during muscle maturation. Nature, Lond. 218, 962-964. LEBErmV A. V. (1971) Nitrogenous extractive compounds of muscle tissue of invertebrates. Jurnal evoljuzionnoj Biokhimii i Physiologii (USSR). (In press.) LEBEDEV A. V. & BOLDY~V A. A. (1972) Precursors of histidine containing dipeptides in muscle tissue of invertebrates. Biokhimija (USSR). 37, 1. LoE P.-R. & FLOm~YE. (1966) The distribution of acetylcholine and cholinesterase in the nervous system and in innervated organs of Octopus dofleini. Comp. Biochem. Physiol. 17, 509-522. MOORE S., SPACKMAND. H. & STEIN W. H. (1958) Chromatography of amino acids on sulfonated polystyrene resins. Analyt. Chem. 30, 1185-1190. SEVERIN S. E. (1967) The participation of natural imidazole containing dipeptides in biosynthesis and reception of acetylcholine. Uspekhi sovremennoj biologii (USSR). 64, 181-196. SEVERIN S. E. & FEDOROVAV. N. (1952) Content of carnosine, anserine, histidine and fl-alanine in skeletal muscle of hen in embrionic development. Dokl. Acad. Nauk Sci. S S S R 82, 443-446. VULPHSONP. L. (1962) Comparative biochemical analysis of amino acids and dipeptides in muscle tissue. Uspekhi biologieheskoj khimii (USSR). 4, 82-92. WOOD J. D. (1958) Nitrogen excretion in some marine teleosts. Can. J. Biochem. Physiol. 36, 1237-1242.
Key Word Index--Histidine dipeptides; carnosine; anserine; cholinergic; Helix pomatia ; Anodonta cygnea ; molluscs; Ommastrephes pacificus.