Download Origins and Evolution of Pathways of Anaerobic Metabolism in the

AMER. ZOOL., 31:522-534 (1991)
Origins and Evolution of Pathways of Anaerobic
Metabolism in the Animal Kingdom1
DAVID ROBERT LIVINGSTONE
Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, United Kingdom
SYNOPSIS. Energetic characteristics and functional roles define two main types of anaerobic
pathways in the animal kingdom: high efficiency/low rates of energy production pathways
geared to anoxia survival (aspartate-succinate and glucose-succinate pathways), and low
efficiency/high rates of energy production pathways geared to maintaining or increasing
metabolic activity (multiple opine pathways and lactate pathway). The aspartate-succinate
and opine pathways require both amino acids and carbohydrate as substrates, whereas the
glucose-succinate and lactate pathways are dependent on carbohydrate only. Phylogenetic,
functional and chemical considerations indicate an evolutionary progression from amino
acid-linked to carbohydrate-based anaerobic pathways. The tauropine and strombine pathways are possibly the most ancient opine pathways so far discovered, and the octopine
pathway the most advanced. The roles of the aspartate-succinate and opine pathways may
originally have been not too dissimilar. A hierarchy of "rates of energy production pathways"
of phosphagen > lactate > octopine > other opine pathways is proposed, which defines
much of their phylogenetic selection and how they are used. The different properties of
phosphocreatine compared to other phosphagens is indicated to have been a key factor in
the emergence of vertebrates.
INTRODUCTION
In a previous paper (Livingstone, 1983),
the evolution of anaerobic metabolism in
animals was considered from the viewpoint
of the functional nature of the pathways and
the interaction of this with the physical and
biological aspects of the environment. A
review of anaerobic metabolism in the
invertebrate and vertebrate phyla identified
four pathway-types of interest: the lactate
and opine pathways used for maintaining
or increasing metabolic activity, and the
glucose-succinate and aspartate-succinate
pathways used for anoxia survival. Based
on the phylogenetic distribution of these
pathways, and other considerations, a hypothetical scheme of the evolution of anaerobic pathways was proposed which indicated early presences for amino acid-based,
opine and aspartate-succinate pathways. A
consideration of chemical, fossil and functional approaches to the reconstruction of
the beginning of life suggested an early role
for opine pathways in providing energy for
the burrowing of infaunal worms of the Precambrian era.
In this paper, I examine these and other
proposals in the light of recent theories of
biochemical evolution, and the substantial
amount of information since published on
the quantitative aspects and energetic characteristics of anaerobic metabolism, including the discovery of new pathways. Also
considered are phosphagens, metabolic acidosis and constraints that operate on biochemical evolution.
ANAEROBIC PATHWAYS AND PHOSPHAGENS
Pathway types and characteristics
The structural, functional and energetic
characteristics of anaerobic pathways in
animals have been extensively analyzed (see
references below for details of pathways).
Various stoichiometric/redox-balance combinations of oxidation (carbonyl to hydroxyl,
aldehyde to carboxyl), reduction (carbonyl
to hydroxyl, double bond saturation, reductive condensation) and energy providing
(substrate level and electron transfer level
phosphorylations) reactions result in the
many different anaerobic pathways seen
across the animal kingdom (Fields, 1988).
Carbohydrate with its multiple hydroxyl
groups for coupled oxidoreduction reactions is an ideal storage substrate, whereas
the use of proteins and amino acids as sub1
From the Symposium on The Origin and Evolution strates is limited by the lack of hydroxyl and
of Metabolic Pathways in Animals presented at the presence of carboxyl groups: fatty acids repAnnual Meeting of the American Society of Zoologists,
resent the limit of carbohydrate fermenta27-30 December 1989, at Boston, Massachusetts.
522
EVOLUTION OF ANAEROBIC METABOLISM
tion (i.e., maximum reduced state) and as
an end-product have the advantage of passing most easily through lipid membranes
(Fields, 1988). Branched pathways with
multiple end-products (glucose-succinate
and aspartate-succinate pathways) produce
low rates of energy output at relatively high
efficiencies (5 to 7 ATP molecules per glucose unit) for environmental anaerobiosis,
e.g., aerial exposure, parasitism, whereas
linear (lactate pathway) and semi-linear
(opine pathways) pathways with single endproducts produce high rates of energy outputs at low efficiences (3 ATP molecules per
glucose unit) for functional anaerobiosis,
e.g., exercise, recovery from anoxia (Livingstone, 1982; Ellington, 1983; Gade,
1983a; De Zwaan and Putzer, 1985; De
Zwaan and Van den Thillart, 1985). The
aspartate-succinate and opine pathways
have in common a requirement for both
carbohydrate and amino acids as substrates,
whereas the glucose-succinate and lactate
pathways are dependent on carbohydrate
only. The rate of substrate consumption is
much greater for the low efficiency/high rates
of energy production than for the high efficiency/low rates of energy production pathways. Anoxia survival is generally characterized by a marked decrease in total rates
of energy production, whereas anaerobic
muscular activity involves a marked
increase. Switching from aerobic metabolism to low or high output modes of anaerobic energy production generally requires,
respectively, a decrease and marked increase
in glycolytic flux (De Zwaan and Van den
Thillart, 1985). Thermodynamic considerations indicate that the different anaerobic
pathways can be designed for economy
(energetic efficiency) or power (high rates of
energy production), but not for both
(Gnaiger, 1983).
The aspartate-succinate and glucose-succinate pathways are closely related pathways, often found coupled together in the
same tissues (De Zwaan and Putzer, 1985).
Although the functions of the pathways have
been considered similar in both providing
low rates of energy production during environmental anaerobiosis (Livingstone, 1983),
differences are evident, indicative of the
aspartate-succinate pathway being used in
523
situations, or tissues, where relatively higher
rates of energy production are required.
Where the two pathways occur together, the
aspartate-succinate pathway functions during the early stages of environmental anaerobiosis, whereas the glucose-succinate
pathway does not become operative until
later. A regulatory role for aspartate, via
its activation of pyruvate kinase (E.C.
2.7.1.40), has been proposed in anoxia tolerant molluscs (Storey, 1986): a depletion
of aspartate, and inhibition of pyruvate
kinase, would presumably favour carbon
flow through the glucose-succinate pathway. Phosphagen is also used during the
early stages and the total rates of energy
production, although reduced from aerobic
rates, are greater than in the later stages, e.g.,
the total rates in fimol ATP min"1 g~' wet
weight for four species of marine bivalve
(Mytilus edulis, Cardium edule, Geukensia
demissa granosissima and Lima hians) were
0.021 to 0.121 in the first four hours of anaerobiosis compared to 0.005 to 0.021 after
ten hours (De Zwaan and Putzer, 1985). The
glucose-succinate pathway is found in most,
if not all, tissue types, whereas the aspartatesuccinate pathway is typical of active tissues, such as adductor muscle and heart tissue of bivalves, which are characterized by
high aspartate pools (De Zwaan and Putzer,
1985). The aspartate-succinate pathway in
the posterior adductor muscle of the common mussel M. edulis is used during hypoxia when the response of the whole animal
is to try and maintain metabolic rates (Bayne
and Livingstone, 1977; Livingstone and
Bayne, 1977). During the early stages of
environmental anaerobiosis, in various
marine invertebrates, the formation of
opines and lactate is minimal and the aspartate-succinate pathway is argued to outcompete the glycolytic end-product pathways by
virtue of its greater affinity for NADH (De
Zwaan and Putzer, 1985). The utilization of
aspartate usually exceeds the production of
succinate through its oxidative decarboxylation to alanine via malic enzyme (E.C.
1.1.1.40) and transaminase reactions, and
this pathway has been proposed to functionally replace the well-known aspartatemalate shuttle in rapidly transferring reducing equivalents into the mitochondria
524
DAVID ROBERT LIVINGSTONE
during environmental anaerobiosis (De
Zwaan and Putzer, 1985; De Zwaan and
Van den Thillart, 1985). All of these features are indicative of a pathway suited for
shorter rather than longer term anaerobiosis, when reduction of metabolic rate is
less than later, and the intrinsic scope for
ATP power output of the aspartate-succinate pathway has been proposed to be greater
than that of the glucose-succinate pathway
(De Zwaan and Putzer, 1985). The use of
the aspartate-succinate pathway is presumably limited by the size of the free aspartate
pool, i.e., equals low capacity relative to glycogen pool (De Zwaan and Putzer, 1985).
A functionally equivalent pathway to the
aspartate-succinate pathway in freshwater
and terrestrial invertebrates, lacking substantial free amino acid pools, is thought to
be the malate-succinate pathway, e.g., malate levels are high in the midge Chaoborus
crystallinus and earthworm Lumbriculus
variegatus and are converted to succinate
during environmental anaerobiosis (De
Zwaan and Putzer, 1985; De Zwaan and
Van den Thillart, 1985).
The lactate pathway is primarily a high
rate of energy production pathway for functional anaerobiosis whose usefulness for
anoxia tolerance is very limited. Some longterm survival is seen with various vertebrate species which possess a lactate pathway only, but this is usually associated with
hypoxic rather than anoxic conditions, and
with a significant depression of metabolic
rate (De Zwaan and Van den Thillart, 1985).
Lactate is formed as a major end-product
during environmental anaerobiosis in nonmarine invertebrates such as gastropods and
parasitic platyhelminths and nematodes, but
often, although not always, in conjunction
with the simultaneous operation of the glucose-succinate pathway (Livingstone, 1982,
1983). Some lactate is also formed during
recovery from anoxic conditions, e.g., in
various crustaceans (De Zwaan and Putzer,
1985). L-lactate is formed by vertebrates
and higher invertebrates such as crustaceans
and echinoderms, e.g., the crab Potamon
warreni (Van Aardt and Wolmarans, 1987)
and the sea urchin Echinus esculentus (Spicer et al., 1988), whereas D-lactate is typical
of molluscs, e.g., the ormer Haliotis lamel-
losa (Gade, 1988) and certain other groups
such as arachnids, e.g., the spider Filistata
hibernalis (Prestwich, 1988a, b). The
importance of the lactate pathway in providing high rates of energy production for
muscular activity and locomotion, and for
other forms of maintained or increased metabolic activity, is reflected in the direct scaling of lactate dehydrogenase (LDH; E.C.
1.1.1.27 and 1.1.1.28) activities with body
mass in fish (Somero and Childress, 1980)
and other vertebrates (Hochachka et al.,
1988), and possibly with its apparent selection in the active and evolutionarily advanced nereid polychaetes and the group of
bivalves (Cardium sp. and Anodonta cygnea) whose metabolic rate is not drastically
reduced during aerial exposure (Livingstone, 1982; Livingstone et al., 1983).
The opine pathways are functionally
analogous to the lactate pathway, at least in
energetic terms, but the maximal rates of
energy production that might be realised
from these pathways are argued to be less
than the lactate pathway (Livingstone,
1983). The terminal reaction of the opine
pathway involves the reductive condensation of pyruvate with an amino acid to form
an imino acid derivative (opine), viz.:
pyruvate + amino acid + NADH + H +
= opine + H2O + NAD +
The major opine pathways so far characterized involve reductive condensation of
pyruvate with arginine, alanine or glycine
to produce, respectively, octopine, alanopine and strombine. The substrate specificities of the enzymes catalyzing the terminal
reactions vary, but they are generally known,
respectively, as octopine dehydrogenase
(ODH; E.C. 1.5.1.11), alanopine and strombine dehydrogenases (ADH and SDH). The
characteristics of the opine pathways have
been extensively studied and the enzyme
activities are highest in muscular tissues
(Livingstone, 1982; Ellington, 1983; Gade,
1983a; De Zwaan and Dando, 1984; De
Zwaan and Putzer, 1985; Gade and Grieshaber, 1986). More recently, additional
opine pathways have been discovered, viz.
tauropine and /3-alanopine formed, respectively, from taurine and /3-alanine through
the actions of tauropine and /3-alanopine
EVOLUTION OF ANAEROBIC METABOLISM
dehydrogenases (TDH and BDH) (Gade,
1986, 1988; Sato et al, 1987; Doumen and
Ellington, 1987), and there is the possibility
that others may exist, utilizing other free
amino acids.
Opine pathways are predominantly
employed during exercise, e.g., tauropine in
the shell adductor muscle of H. lamellosa
(Gade, 1988), and recovery from exercise
or anoxia, e.g., octopine in the phasic
adductor muscle of the scallop Placopecten
magellanicus (Livingstone, 1982). The formation of strombine in the posterior adductor muscle of M. edulis is related to valve
opening and closing (De Zwaan and Dando,
1984; Shick et al, 1986) and was significantly enhanced when movement of the
shells was prevented (De Zwaan et al, 1983).
Multiple opines can be formed and different
opines can be formed under different conditions, e.g., alanopine and strombine were
formed in the body wall musculature of the
lugworm Arenicola marina, the former predominating during exercise (ratio of 6:1) and
the latter during anoxia (ratio of 1:3.5) (Siegmund et al, 1985). Minor amounts of opines
are formed during environmental anaerobiosis, e.g., octopine and alanopine/strombine in the posterior adductor muscle of the
bivalve Scapharca inaequivalvis (Isani et al,
1989), and (in decreasing proportions)
/3-alanopine, strombine, tauropine and alanopine in adductor muscle of the blood shell
Scapharca broughtonii (Sato et al, 1988).
Various energetic functions have been proposed for opine formation during recovery
from environmental or functional anaerobiosis, including physical (valve) movements, maintenance of metabolic rates
(Livingstone, 1982), and supplementing
normal (or elevated) aerobic ATP yielding
processes involved in recharging of phosphagen and ATP pools, resynthesis of
aspartate and glycogen, clearance of acidic
end-products and restoration of normal
physiological activities such as feeding (De
Zwaan and Putzer, 1985; Shick et al, 1988).
Based on enzyme kinetics and other observations, it has been argued that the octopine
pathway is likely to realise higher rates of
energy production than either the strombine
or alanopine pathways (Livingstone et al,
1983).
525
Other anaerobic pathways exist in addition to these four major types. Ethanol formation is used as a means to limit metabolic
acidosis in certain anoxia tolerant fish species, e.g., goldfish Carassius auratus (De
Zwaan and Van den Thillart, 1985). Discrepancies between theoretical anoxic energy
output (calculated from accumulated fermentation products) and that measured
directly by calorimetry has suggested the
existence of unidentified anaerobic pathways, e.g., accumulated end-products in
whole M. edulis (Shick et al, 1988) and isolated ovaries of the sea urchin Strongylocentrotus droebachiensis (Bookbinder and
Shick, 1986) accounted for only, respectively, 50% and 37% of total anoxic heat
dissipation. However, similar "exothermic" gaps are also seen for vertebrate species (see Shick et al, 1988) and it may be
they reflect phenomena other than endproduct accumulation.
Phosphagens and a hierarchy of rates
of energy production pathways
A second important source of anaerobic
energy are the various phosphoguanidine
compounds, or phosphagens, which are
transphosphorylated by specific phosphokinases to yield ATP according to the reaction:
phosphoguanidine + MgADP + H +
= guanidine + MgATP
Phosphagens are used during both muscular
activity and anoxia survival, e.g., for a number of species of mollusc, total rates of
anaerobic energy production (mean ± SEM
in jtmol min~' g~' wet wt.; n = 5 to 12)
during exercise and short-term anaerobiosis
were, respectively, 6.4 ± 1.7 and 0.044 ±
0.007, of which, respectively, 55.4 ± 5.2%
and 50.0 ± 10.6% were provided by the
phosphagen (calculated from Table 1 of De
Zwaan and Van den Thillart, 1985). Maximum rates of energy production from
phosphagens generally exceed those of the
lactate or opine pathways (Table 1), and,
possibly for this reason, phosphagen utilization during exercise often precedes, to
varying degrees, the accumulation of glycolytic end-products. This tendency is
observed across the animal kingdom, e.g.,
526
DAVID ROBERT LIVINGSTONE
TABLE 1. Maximum observed rates of energy production (in y.mol min~' g-' wet weight) during exercise (or
recovery from anaerobiosis*) from phosphagen and lactate or opine anaerobic pathways in vertebrates and invertebrates.
Organism
Phosphagen breakdown
Man
Other mammals
Reptile
Amphibian
Fish
Arachnid
Crustacean
Annelid
Mollusc
96-360
16.9
—
37.1
12.0
41.3 ± 10 (3)
20.0 ± 2.5 (3)
0.55 ± 0.5 (2)
3.6 ± 0 . 8 (21)
Anaerobic pathway
Lactate
Lactate
Lactate
Lactate
Lactate
Lactate
Lactate
Lactate
Octopine
Octopine*
Strombine/alanopine
Strombine/alanopine*
Strombine*
Alanopine*
Tauropine
Anaerobic energy
References
60-210
5.1 ± 3 (3)
16.1 ± 4 (3)
6.9 ±2.1 (4)
8.4 ± 3.4(8)
9.9 ± 3.1 (3)
2.9 ± 1.6(3)
0.8 ± 0.3 (5)
1.4 ±0.5 (8)
0.39 ± 0.2 (3)
0.28 ±0.1 (4)
0.12
0.03 ± 0 (3)
0.03
0.46
1,2
3
3,4
3,5
3,6
7,8
9
9, 10
3, 9, 11, 12
12
11
11
13, 14
13
15
Data presented are comprehensive for marine invertebrates, but illustrative for other animal groups. Maximum
rates were calculated for each species and results presented as single values, means ± range (n = 2) or ±SEM
(number of species given in parentheses). Rates are generally for muscular tissues although some whole animal
data were used. For these and other details see references, viz., 1: Hochachka (1985); 2: Livingstone (1982); 3:
De Zwaan and Van den Thillart (1985); 4: Gleeson and Dalessio (1989); 5: Miller and Sabol (1989); 6: Dalla
Via et al. (1989); 7: Prestwich (1988a); 8: Prestwich (19886); 9: De Zwaan and Putzer (1985); 10: Siegmund et
al. (1985); 11: Baldwin and England (1982); 12: Livingstone (1982); 13: Eberlee et al. (1983); 14: De Zwaan et
al. (1983); 15: Gade (1988).
in spiders (Prestwich, 19886), molluscs,
crustaceans and fish (De Zwaan and Van
den Thillart, 1985). Phosphagen concentrations are highest in muscular tissues and
many observations testify to their importance in high energy demanding activities,
e.g., exhaustion in scallops, decreased
sprinting speeds in spiders and reduced
escape responses in gastropods coincide with
phosphagen depletion (De Zwaan and Van
den Thillart, 1985; Prestwich, 19886).
Phosphagen is the main energy source for
contraction of isolated anterior byssus
retractor muscle of M. edulis, with octopine
formation being invoked with increasing
energy demand (Zange et al., 1989).
A hierarchy of rates of energy production
pathways of lactate > octopine > alanopine
and strombine has been proposed based on
observed maximum rates of energy production, pathway design and enzyme kinetics (Livingstone, 1982, 1983; Livingstone #
al., 1983). To the top of this list, with the
greatest intrinsic potential for high rates of
energy production, can be added the phosphagens (Table 1). Although many factors
will influence rates of energy production, the
comparisons between the pathways hold
within the different animal groups, and, to
some extent, it could be argued between
them, e.g., the lactate pathway in crustaceans and arachnids compared to the octopine pathway in molluscs. Differences are
also seen between the phosphagens, the relative equilibrium constants for phosphagen
formation being lower for phosphoarginine,
phosphoglycocyamine, phosphotaurocyamine and phospholombricine than for
phosphocreatine, i.e., the latter is thermodynamically less stable (Ellington, 1989).
Information on the newly discovered
^-alanopine and tauropine pathways is limited, but observed rates of energy production (Table 1) and the high apparent K,,,
values for amino acid substrate of the dehydrogenases (Gade, 1986; Doumen and
Ellington, 1987) indicate their energetic
potential is similar to that of the strombine
and alanopine pathways. At the bottom of
the rates of energy production pathways list
can be placed the glucose-succinate pathway
designed for energetic efficiency and anoxia
survival, e.g., 0.008 to 0.13 ^mol min"1 g~'
wet weight for muscular tissue of various
EVOLUTION OF ANAEROBIC METABOLISM
bivalve and gastropod species (Livingstone,
1982).
527
genetic distribution of the lactate and glucose-succinate pathways accords with the
anoxia tolerances of the organisms. The
Phylogenetic distribution
marked presence of the lactate pathway and
The distributions of phosphagens and the nature of the phosphagens in the Choranaerobic pathways across the animal king- data and Echinodermata are consistent with
dom are well characterized, including to a their phylogenetic relationship in the Deureasonable degree the opine pathways (Liv- terostomia. A consistency is also seen in the
ingstone, 1982; Gade and Grieshaber, 1986; phylogenetic relationships between the
De Zwaan and Putzer, 1985; Ellington, Annelida and the Arthropoda, and the
1989). Phosphocreatine and phosphoargi- Lophophorata (Brachiopoda) and the Deunine occur predominantly in, respectively, terostomia, in that the former group of each
vertebrates and invertebrates. The excep- pair contain both opine and lactate pathtions are the Echinodermata, which contain ways, whereas the latter groups have lost
both (the primitive crinoids contain argi- the opine pathways (Livingstone et ai,
nine kinase [E.C. 2.7.3.3] only), and the 1983).
Annelida which contain four other major
The distribution of individual opine
phosphagens in addition to these two, i.e., pathways has been studied in terms of both
phosphoglycocyamine, phosphotaurocy- in vivo end-product formation and, to a
amine, phosphohypotaurocyamine and much greater extent, the presence of specific
phospholombricine. The flagellated, highly dehydrogenase activities. The correlation
motile sperm of echinoderms and poly- between the two is by no means absolute
chaetes contain creatine kinase (E.C. 2.7.3.2) but nevertheless reasonable, e.g., tauropine
only, whereas the eggs of echinoderms and formation in H. lamellosa (Gade, 1988) and
tissues of polychaetes contain arginine /8-alanopine formation in S. broughtonii
kinase or other phosphokinases. The sub- (Sato et ai, 1987,1988) corresponded with,
units of the different kinases often hybridize respectively, high TDH and BDH activities.
together, e.g., sea cucumber Caudina are- The octopine pathway is absent from the
nicola arginine kinase with rabbit brain cre- Polychaeta, characteristic of the Mollusca,
atine kinase (Seals and Grossman, 1988), and present in other marine invertebrate
and the different kinases are thought to have phyla. Octopine formation has been
originated from an ancestral arginine kinase observed in some 15 species of bivalve, gasgene.
tropod and cephalopod mollusc, Sipunculus
The lactate pathway is present to some nudus (Sipuncula) and Cerebratulus lacteus
degree in all phyla, but is the sole major (Nemertina) (Gade, 1983&; De Zwaan and
anaerobic pathway of the higher or evolu- Putzer, 1985; Isani et ai, 1989). Alanopine
tionarily most advanced ones (Chordata, or strombine formation has been observed
Echinodermata, Arthropoda). In contrast, in S. nudus, A. marina (Annelida) and 15
the opine pathways are essentially found in species of bivalves and gastropods, but not
marine species of the lower (Porifera, Cni- in cephalopods (Korycan and Storey, 1983;
daria, Nemertina) and middle (Mollusca, De Zwaan and Putzer, 1985; Isani et ai,
Annelida, Brachiopoda, Sipuncula) phyla. 1989); tauropine and /3-alanopine formaThe glucose-succinate pathway is charac- tion occurs in S. broughtonii and tauropine
teristic of organisms regularly experiencing in the archaeogastropod H. lamellosa (see
anaerobiosis (most lower and middle phyla, before). Consistencies in the phylogenetic
including the parasitic Nematoda and distribution of opine dehydrogenase activPlatyhelminthes), and is also found in insect ities are observed down to the level of phyla,
larvae. The aspartate-succinate pathway class, order and even family (Table 2). SDH
occurs in molluscs and polychaetes, and is characteristic of the Porifera, and ODH
minor presences of the succinate pathways and ADH are high in the Cnidaria. ODH is
are found in crustaceans and vertebrates (De the sole major dehydrogenase activity of
Zwaan and Van den Thillart, 1985; Van some of the more active molluscs, i.e.,
Aardt and Wolmarans, 1987). The phylo- Cephalopoda, Scaphopoda and swimming,
528
DAVID ROBERT LIVINGSTONE
TABLE 2. Relative pyruvate oxidoreductase activities in muscular or whole tissues of various marine invertebrate
groups.
Phyla, class, order
or family
Porifera
Cnidaria
Nemertina
Brachiopoda
Annelida
Polychaeta
Mollusca
Polyplacophora
Gastropoda
Archaeogastropoda
Mesogastropoda
Neogastropoda
Bivalvia
Myidae
Ostreidae
Veneridae
Mytilidae
Tellinidae
Cardiidae
Pectinidae
Scaphopoda
Cephalopoda
Crustacea
Echinodermata
Dehydrogenases
Number
of species
Lactate
Octopine
Alanopine
Strombine
6
10
3
3
0.41 (0.20)
0.23(0.10)
0.44 (0.29)
0.02 (0.02)
C). 17 (0.17)
C).70(0.15)
C).67 (0.33)
0.03 (0.03)
0
(0)
0.52(0.12)
0.25(0.10)
0.83(0.13)
0.57 (0.20)
0.29(0.11)
0.09 (0.06)
0.71 (0.29)
15
0.31(0.11)
0.07(0.07)
0.54(0.11)
0.38(0.12)
4
29
9
10
10
50
2
5
10
5
3
2
3
1
10
11
8
1.0 (0)
0.40 (0.08)
0.47(0.12)
0.53(0.14)
0.07 (0.02)
0.21(0.05)
1.0 (0)
0.03 (0.02)
0.08 (0.02)
0.18(0.13)
0.07 (0.03)
0.34 (0.33)
0
(0)
0.28
0.05 (0.02)
1.0 (0)
1.0 (0)
0.04(0.02)
().33 (0.08)
0.11(0.07)
(). 15 (0.10)
().67(0.13)
0.59(0.06)
() (0)
C) (0)
0.44 (0.09)
C).76(0.17)
.0 (0)
0.01 (0.01)
0.54 (0.08)
0.46(0.16)
0.53(0.11)
0.63(0.12)
0.43 (0.06)
0
(0)
0.90 (0.03)
0.86 (0.06)
0.42(0.15)
0.14(0.03)
0.12(0.06)
0.03 (0)
0.01
0
(0)
0
(0)
0
(0)
0.02 (0.02)
0.15(0.04)
0.12(0.08)
0.24 (0.08)
0.15(0.04)
0.46 (0.06)
0
(0)
0.97 (0.02)
0.96 (0.02)
0.40(0.11)
0.12(0.03)
0.19(0.05)
0.03(0.01)
0
0
(0)
0.06 (0.05)
0.03 (0.02)
•0 (0)
.0 (0)
.0
.0 (0)
() (0)
().07 (0.06)
For each species the highest dehydrogenase specific activity (in activity g~' wet weight) was given a value of
1 and the others calculated relative to this. Means and SEM were than calculated for each dehydrogenase activity
for each particular group of animals. SEM given in parentheses. Original data taken from Baldwin (1982),
Baldwin and England (1982), Eberlee et al. (1983), Kluytmans et al. (1983), Korycan and Storey (1983), Livingstone et al. (1983), Bowen (1987), Sato et al. (1987) and D. R. Livingstone, W. B. Stickle, M. Kapper, S.
Wang and W. Zuburg (unpublished). Activities were mainly for muscular tissues, although in some cases whole
animals were used.
burrowing and jumping bivalves (Pectinidae, Solenidae, Cardiidae and Tellinidae).
ODH is prominent in advanced Neogastropoda, whereas ADH features in this gastropod order, the Mesogastropoda and the
more primitive Archaeogastropoda. Specific activities of ADH (not shown) are much
lower in Archaeogastropoda than in the
other two gastropod orders (Livingstone et
al., 1983). Much less is known of the distribution of the recently discovered TDH
and BDH activities: BDH is prominent in
S. broughtonii, whereas TDH appears characteristic of Archaeogastropoda and the
ancient Brachiopoda (TDH activity in pedicle of Glottidea pyramidata was about x 2
that of ADH) (Doumen and Ellington, 1987;
Sato et al., 1987; Gade, 1988; Hammen,
1989). TDH or BDH could possibly be pres-
ent in certain marine groups lacking ADH,
SDH and ODH, accounting for the apparent
absence of opine dehydrogenase activities,
viz., in the primitive Polyplacophora and
possibly in the Myidae and Nudibranchia
(Table 2).
EVOLUTION OF ANAEROBIC PATHWAYS
AND PHOSPHAGENS
The earliest environmental conditions and
organisms were anaerobic, the transition to
various degrees of oxygen-based existence
occurring between some 2,700 and 1,600
million years ago with the appearance of,
respectively, the photosynthetic blue-green
algae and the first aerobes: key biochemical
pathways are proposed to have arisen in this
order—rudiments of glycolysis, electron
transport chain, polyphosphate phospha-
EVOLUTION OF ANAEROBIC METABOLISM
gens, arginine phosphate and creatine phosphate (Fox, 1988). The first discernible
invertebrates (Porifera) occurred some 700
million years ago, followed by the appearance of most major invertebrate phyla
around 700 to 570 million years ago (Precambrian/Cambrian border). Oxygen was a
key factor in the development of metazoan
life through its requirement for hydroxyproline, hydroxylysine and resultant collagen synthesis (Towe, 1981).
Anaerobic pathways
An hypothetical scheme for the evolution
of anaerobic pathways is given in Figure 1.
An early existence for rudiments of the glycolytic pathway is indicated (Fox, 1988),
and it is interesting to speculate whether the
glucose-succinate pathway is a modification
of the later evolved Krebs cycle, or whether
the reverse occurred and the rudimentary
reactions of the anaerobic pathways that
gave rise to the glucose-succinate pathway
also gave rise to the Krebs cycle. A number
of observations support the proposal for the
early appearance of amino acid based pathways. Amino acids were prominent components of the primaeval soup, and the relative abundance of the four major ones
present in various cosmic environments
(moon, meteorites, hot terrestrial lava) and
formed in laboratory simulations (reducing
atmosphere and electric discharge, heating)
is exactly the same, viz., in order of decreasing abundance, glycine, alanine, glutamic
acid and aspartic acid (Fox, 1988). The next
two to five most abundant amino acids vary
for the different sources and include proline
(required for hydroxyproline synthesis), but
not arginine (required for phosphoarginine
and octopine synthesis). Glycine, alanine
and aspartate are utilized, respectively, by
the strombine, alanopine and aspartate-succinate pathways: /3-alanine is an end-product of pyrimidine metabolism and can also
be derived from the decarboxylation of
aspartate. Protein, not carbohydrate, is
indicated to be the major substrate for anoxic
energy metabolism in lower organisms, such
as coelenterates (Shick et ah, 1988), and in
the juvenile forms of others, such as larvae
of the oyster Crassostrea virginica (Widdows et al, 1989). The major anaerobic end-
529
products of coelenterates are alanine and
glutamate, e.g., in the sea anemone Actinia
equina (Navarro and Ortega, 1984). The use
of the aspartate-succinate pathway is thought
to have a sparing effect on what carbohydrate stores there may be (De Zwaan and
Putzer, 1985), and ammonia production
during anaerobiosis can be used for acidbase balance (Shick et al, 1988). A phylogenetic and functional line of consistency
therefore appears to exist for an early prominence for amino acids in anaerobic metabolism.
The divergence of the amino acid pathways into the aspartate-succinate and opine
pathways resulted in the appearance and
subsequent evolution of the two main types
of anaerobic pathway geared to, respectively, anoxia survival and maintaining or
increasing metabolic activity. The functional distinction between the two types may
initially not have been great, given the energetic features of aspartate-succinate pathway and the fact that both types of pathways
would be similarly limited by the size of
their free amino acid pools (also see below).
However, with selection for particular features such as ATP yield and decreased or
increased glycolytic flux, their roles would
have become better denned. Correlations
between the size of free amino acid pools
and the presence of opine pathways are seen
in modern day species, e.g., /3-alanine in S.
broughtonii (Sato et al., 1987), and presumably this must have been a factor in their
selection. Multiple opine pathways increase
the capacity of the system, i.e., the size of
the amino acid pool is effectively the sum
of components, and have been argued to
guarantee continuous glycolytic flux during
conditions of exercise or anoxia/hypoxia
followed by recovery (Gade and Grieshaber, 1986). Strombine and tauropine may
be the oldest opine pathways, a major presence being indicated in, respectively, the
primitive Porifera and the ancient Brachiopoda (the status of the tauropine pathway
in the Porifera and the other lower phyla is
unknown). Glycine and taurine are often the
largest free amino acid pools found in species, and this could have given the opine
pathways an early advantage over the aspartate-succinate pathway in functional an-
530
DAVID ROBERT LIVINGSTONE
aerobiosis, or even allowed them to have a
role in anoxia survival, i.e., generating a low
rate of energy production over a long period
of time.
The selection of the octopine pathway in
active molluscs, including the evolutionarily
advanced cephalopods and neogastropods,
may have been linked to its capability for
higher rates of energy production and/or its
relationship with the molluscan phosphagen, phosphoarginine. Phosphoargine
breakdown provides arginine for octopine
formation, so freeing it from the constraints
of any interaction with other aspects of
amino acid metabolism, although integration between the two processes in modern
day molluscs is generally minimal (De
Zwaan and Putzer, 1985). The octopine
pathway is usually found associated with
high phosphoarginine pools, and is the only
opine pathway found in freshwater invertebrates (bivalves) (De Zwaan and Putzer,
1985). A novel origin for the octopine pathway in molluscs from bacterial transfection
has been suggested (Hochachka, 1988), but
if this is so, then, given the wide phylogenetic distribution of the pathway (Nemertina, Sipuncula) and/or ODH activity (Table
2), it must have been a general phenomenon
and occurred early in the evolution of the
Animalia.
The advantages of carbohydrate over
protein and amino acids as an anaerobic
substrate would have been a major factor
in the selection of the glucose-succinate and
lactate pathways. Movement to freshwater
and terrestrial existences would also have
contributed to the loss of the amino acidlinked pathways from the lower and middle
phyla. The possible relationships between
the aspartate-succinate and glucose-succinate pathways have been discussed before
(Livingstone, 1983). The lactate pathway
could have originated independently of the
opine pathways, or possibly from them. An
evolutionary relationship (progression)
between monomeric opine dehydrogenases,
dimeric D-LDH and tetrameric L-LDH has
been speculated at (Livingstone et ai, 1983)
(N.B., a monomeric LDH, is present in
primitive fish but a tetrameric LDH is found
in ascidians—Baldwin, 1988). The functions of the pathways would have become
HIGH EFFICIENCY / LOW RATES LOW EFFICIENCY / HIGH RATES
OF ENERGY PRODUCTION
OF ENERGY PRODUCTION
PATHWAYS
PATHWAYS
FIG. 1. Hypothetical scheme of the evolution of the
pathways of anaerobic metabolism (modified from
Livingstone, 1983).
increasingly distinct by selection for their
key features, viz. fuel stores, ATP yield or
rates on energy production, levels and regulation of glycolytic enzymes, and mechanisms for dealing with metabolic acidosis,
such as excretable (volatile) end-products
and others (see below) (De Zwaan and
Putzer, 1985; Hochachka, 1985; Hochachka
et ai, 1988). For example, the magnitude
of the Pasteur effect (increased glycolytic flux
during functional anaerobiosis) increases
through the Mollusca, Crustacea and Vertebrata (De Zwaan and Putzer, 1985; De
Zwaan and Van den Thillart, 1985), e.g.,
x 50 for swimming bivalves compared to
x 2,300 for man (Livingstone, 1983). Similarly, total rates of ATP turnover during
anoxia are reduced about x 75 in molluscs
compared to xlO in crustaceans, so contributing to the prolonged survival of the
former (De Zwaan and Putzer, 1985). In
addition, selection for other features would
have occurred, such as enzyme polymorphism for organ specialization, e.g., see Gade
and Grieshaber (1986) and metabolic efficiency, e.g., allozyme heterozygosity in P.
magellanicus is correlated with increased
octopine formation and may be related to
the scallop's scope for movement (Volckaert and Zouros, 1989).
Phosphagens
The appearance of phosphagens allowed
the storage of energy for subsequent use during periods of high energy need. The change
EVOLUTION OF ANAEROBIC METABOLISM
531
from phosphoarginine to phosphocreatine for the prolonged burrowing of infaunal
is argued to be a central factor in the appear- worms of the Precambrian era (Livingstone,
ance of chordates, providing energy not only 1983). A flagellate ancestor is generally
for increased motility during, for example, assumed for the metazoans, but the status
embryological development, but also driv- of the proposed ancestoral acoelomate (see
ing the transition from rudimentary to higher Livingstone, 1983) {i.e., primitive or seclevel nervous activity (Fox, 1988). The spe- ondarily derived from a coelomate condicific presence of phosphocreatine in highly tion) is a matter of debate (Barnes, 1983).
motile polychaete and echinoderm sperm is Phosphagens would have been present for
presumably testimony to its energetic nervous activity and no doubt also been
advantages. A mechanistic basis for this important in burrowing. The aspartate-sucadvantage is provided by the lower ther- cinate pathway could have made a contrimodynamic stability of phosphocreatine, bution to functional anaerobiosis, but in the
allowing the maintenance of higher ATP/ longer term the opine pathways would have
ADP ratios, necessary for flagellar protein predominated. Movement would have
movement of sperm and rapid contraction/ intensified animal-environmental and anirelaxation of vertebrate muscle, i.e., the mal-animal interactions (Fox, 1988), and
cycles of contraction are promoted by the allowed the invasion of new aerobic and
rapid dissociation of ADP away from spe- anaerobic habitats. Different anaerobic
cific proteins (Ellington, 1989). A selective strategies could have been employed at difadvantage is also conveyed on the other ferent stages of an animal's lifecycle, e.g.,
phosphagens by virtue of their greater ther- anaerobic energy output decreases and
modynamic stability, making them better anoxia tolerance increases of Crassostrea
buffers of ATP levels under the conditions virginica larvae with developmental stage
of reduced pH that occur during long-term and size, indicating a switch from functional
anoxia survival (Ellington, 1989). The phe- to environmental-type anaerobic pathways
nomenon of pluriphosphagens (more than (Widdows et ai, 1989). The combined, or
one phosphagen in one tissue) could be due complementary, use of aerobic and anaerto functional compartmentalization, or obic pathways to create new niches would
involve their sequential use during energy be accompanied by an increase in their integrated control (Simon et ai, 1978). Other
deficit (Ellington, 1989).
aspects of the evolutionary interaction and
The phosphagens and anaerobic path- biochemistry and biology have been disways are used together, or sequentially, to cussed elsewhere (Livingstone, 1983; Livmeet the particular energy demands of the ingstone et ai, 1983).
organism. The scope for energy output is
generally increased up the phylogenetic tree,
e.g., total energy output during functional Constraints on the evolution of
anaerobiosis in various species of mollusc anaerobic pathways
and crustacean was raised, respectively,
The speed and direction of evolutionary
about x20 and x60 (De Zwaan and Van change are continually influenced by selecden Thillart, 1985). Phosphagens can pro- tive constraints operating at various levels
vide the energy crucial for nerve and other of biological organization: the past history
excitable tissue function, whereas anaerobic of evolutionary change may act to constrain
pathways are effective in short- and longer- further adaptive modifications in a manner
term muscular activity.
that may differ between taxonomic groups
simply because of the different evolutionary
Biological considerations
trajectories followed (see Pogson, 1988).
Advances in energy metabolism, regula- Such "local" constraints have been invoked
tion and storage have been hypothesized to to explain the presence of the octopine pathhave been the impetus to phylogenetic way (as opposed to the lactate pathway) in
metamorphosis (Fox, 1988). A prominent the fast-moving Cephalopoda, and the lacrole was assigned to the opine pathways in tate pathway in the slow-moving Echinothe rise of metazoans in providing energy dermata (Livingstone, 1983), although in the
532
DAVID ROBERT LIVINGSTONE
latter case other considerations are possible
(Livingstone et al, 1983). The absence of
the glucose-succinate pathway in the higher
phyla is of interest (Fields, 1988), and could
be due to such constraints, or to the fact
that evolution works to produce energetically efficient but also different animals, i.e.,
with physiologies capable of filling/creating
unique niches.
Acid production is a potential threat to
most cellular activities, and the evolution
of anaerobic pathways was probably closely
linked to the development of mechanisms/
strategies for combating this (Livingstone,
1983). The buffering capacity of muscular
tissues in invertebrates (Eberlee and Story,
1984) and vertebrates (Somero, 1983) is
often related to the capacity for anaerobic
energy production. Mechanisms that have
evolved for limiting metabolic acidosis are
various and include phosphagen hydrolysis;
ammonia production from AMP, e.g., fish,
or amino acids, e.g., coelenterates; mobilization of calcium carbonate, e.g., turtles and
bivalves; small and large buffering molecules, e.g., histidine-containing dipeptides
and increased levels of histidine residues in
enzymes in fish; vasoconstriction to restrict
acid movement, e.g., in fish; neutral or volatile end-products (see before); and different
strategies of substrate resynthesis, e.g., in
the lizard Diposaurus dorsalis (Somero,
1983; De Zwaan and Van den Thillart, 1985;
Gleeson and Dalessio, 1989).
REFERENCES
Baldwin, J. 1982. Correlations between enzyme profiles in cephalopod muscle and swimming behavior. Pac. Sci. 36:349-356.
Baldwin, J. 1988. Adaptation, constraint, and convergence among vertebrate LDH isozymes: An
evolutionary perspective. Can. J. Zool. 66:10111014.
Baldwin, J. and W. R. England. 1982. The properties
and functions of alanopine dehydrogenase and
octopine dehydrogenase from the pedal retractor
muscle of Stromdidae (class Gastropoda). Pac. Sci.
36:381-394.
Barnes, R. D. 1983. Origins of the lower invertebrates. Nature 306:224-225.
Bayne, B. L. and D. R. Livingstone. 1977. Responses
of Mytilus edulis L. to low oxygen tension: Acclimation of the rate of oxygen consumption. J. Comp.
Physiol. 114:129-142.
Bookbinder, L. H. and J. M. Shick. 1986. Anaerobic
and aerobic energy metabolism in ovaries of the
sea urchin Strongylocentrotus droebachiensis. Mar.
Biol. 93:103-110.
Bowen, C. E. 1987. Pyruvate metabolism in Haliotis
(ormer/abalone): Lactate dehydrogenase, pyruvate kinase and opine dehydrogenase activities.
Comp. Biochem. Physiol. 86B:219-226.
Dalla Via, J., M. Huber, W. Wieser, and R. Lackner.
1989. Temperature-related responses of intermediary metabolism to forced exercise and recovery in juvenile Rutilus rutilus (L.) (Cyprinidae:
Teleostei). Physiol. Zool. 62:964-976.
De Zwaan, A. and P. R. Dando. 1984. Phosphoenolpyruvate-pyruvate metabolism in bivalve molluscs. Molec. Physiol. 5:285-310.
De Zwaan, A., A. M. T. De Bont, and J. Hemelraad.
1983. The role of phosphoenolpyruvate carboxykinase in the anaerobic metabolism of the sea mussel Mytilus edulis L. J. Comp. Physiol. 153B:267274.
De Zwaan, A. and V. Putzer. 1985. Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia
to exercise anoxia). In M. S. Laverack (ed.), Physiological adaptations ofmarine animals, SEB Symp.
No. 39, pp. 33-62.
De Zwaan, A. and G. Van den Thillart. 1985. Low
and high output modes of anaerobic metabolism:
Invertebrate and vertebrate strategies. In R. Gilles
(ed.), Circulation, respiration, and metabolism, pp.
167-192. Springer-Verlag, Berlin.
Doumen, C. and W. R. Ellington. 1987. Isolation and
characterization of a taurine-specific opine dehydrogenase from the pedicles of the brachiopod,
Glottidea pyramidata. J. Exp. Zool. 243:25-31.
Eberlee, J. C , J. M. Storey, and K. B. Storey. 1983.
Anaerobiosis, recovery from anoxia, and the role
of strombine and alanopine in the oyster Crassostrea virginica. Can. J. Zool. 61:2682-2687.
Eberlee, J. C. and K. B. Storey. 1984. Buffering capacities of the tissues of marine molluscs. Physiol.
Zool. 57:567-572.
Ellington, W. R. 1983. The recovery from anaerobic
metabolism in invertebrates. J. Exp. Zool. 228:
431^144.
Ellington, W. R. 1989. Phosphocreatine represents a
themodynamic and functional improvement over
other muscle phosphagens. J. Exp. Biol. 143:177194.
Fields, J. H. A. 1988. Fermentative adaptations to
the lack of oxygen. Can. J. Zool. 66:1036-1040.
Fox, R. E. 1988. Energy and the evolution of life. W.
H. Freeman and Co., New York.
Gade, G. 1983a. Energy metabolism of arthropods
and molluscs during environmental and functional anaerobiosis. J. Exp. Zool. 228:415^29.
Gade, G. 19836. Mode of energy production during
environmental and functional anoxia in the
nemertean Cerebratulus lacteus. Amer. Zool. 23:
892.
Gade, G. 1986. Purification and properties of tauropine dehydrogenase from the shell adductor
muscle of the ormer, Haliotis lamellosa. Eur. J.
Biochem. 160:311-318.
Gade, G. 1988. Energy metabolism during anoxia
EVOLUTION OF ANAEROBIC METABOLISM
and recovery in shell adductor and foot muscle of
the gastropod mollusc Haliotis lamellosa: Formation of the novel anaerobic and product tauropine. Biol. Bull. 175:122-131.
Gade, G. and M. K. Grieshaber. 1986. Pyruvate
reductases catalyze the formation of lactate and
opines in anaerobic invertebrates. Comp. Biochem. Physiol. 83B:255-272.
Gleeson, T. T. and P. M. Dalessio. 1989. Lactate and
glycogen metabolism in the lizard Dipsosaurus
dorsalis following exhaustive exercise. J. Exp. Biol.
144:377-393.
Gnaiger, E. 1983. Heat dissipation and energetic efficiency in animal anoxibiosis: Economy contra
power. J. Exp. Zool. 228:471-490.
Hammen, C. S. 1989. Tauropine dehydrogenase in
brachiopods and mollusks. Amer. Zool. 29:159A.
Hochachka, P. W. 1985. Fuels and pathways as
designed systems for support of muscle work. J.
Exp. Biol. 115:149-164.
Hochachka, P. W. 1988. The nature of evolution and
adaptation: Resolving the unity-diversity paradox. Can. J. Zool. 66:1146-1152.
Hochachka, P. W., B. Emmett, and R. K. Suarez.
1988. Limits and constraints in the scaling of
oxidative and glycolytic enzymes in homeotherms. Can. J. Zool. 66:1128-1138.
Isani, G., O. Cattani, E. Carpene, S. Tacconi, and P.
Cortesi. 1989. Energy metabolism and recovery
in the posterior adductor muscle of the bivalve
Scapharca inaequivahis (Bruguiere). Comp. Biochem. Physiol. 93B: 193-200.
Kluytmans, J. H., A. M. T. De Bont, E. C. J. Kruitwagen, H. J. L. Ravestein, and P. R. Veenhof. 1983.
Anaerobic capacities and anaerobic energy production of some Mediterranean bivalves. Comp.
Biochem. Physiol. 75B:171-179.
Korycan, S. A. and K. B. Storey. 1983. Organ-specific
metabolism during anoxia and recovery from
anoxia in the cherrystone clam, Mercenaria mercenaria. Can. J. Zool. 61:2674-2681.
Livingstone, D. R. 1982. Energy production in the
muscle tissue of different kinds of molluscs. In A.
D. F. Addink and N. Spronk (eds.), Exogenous
and endogenous influences on metabolic and neural control, pp. 257-274. Pergamon Press, Oxford.
Livingstone, D. R. 1983. Invertebrate and vertebrate
pathways of anaerobic metabolism: Evolutionary
considerations. J. Geol. Soc. London 140:27-37.
Livingstone, D. R. and B. L. Bayne. 1977. Responses
of Mytilus edulis L. to low oxygen tension: Anaerobic metabolism of the posterior adductor muscle
and mantle tissues. J. Comp. Physiol. 114:143155.
Livingstone, D. R., A. De Zwaan, M. Leopold, and E.
Marteijn. 1983. Studies on the phylogenetic distribution of pyruvate oxidoreductases. Biochem.
Syst. Ecol. 11:415-425.
Miller, K.. and J. L. Sabol. 1989. The role of phosphocreatine in the metabolic support of locomotion by clawed frogs, Xenopus laevis. Comp. Biochem. Physiol. 93B:251-254.
Navarro, E. and M. M. Ortega. 1984. Amino acid
accumulation from glucose during air exposure and
533
anoxia in the sea anemone Actinia equina (L.).
Comp. Biochem. Physiol. 78B: 199-202.
Pogson, G. H. 1988. Constraints on the genetic process of biochemical adaptation. Can. J. Zool. 66:
1139-1145.
Prestwich, K. N. 1988a. The constraints on maximal
activity in spiders. I. Evidence against the fluid
insufficiency hypothesis. J. Comp. Physiol. 158B:
437^147.
Prestwich, K. N. 19886. The constraints on maximal
activity in spiders. II. Limitations imposed by
phosphagen depletion and anaerobic metabolism.
J. Comp. Physiol. 158B:449^t56.
Sato, M., S. Susuki, Y. Yashuda, H. Kawauchi, N.
Kanno, and Y. Sato. 1988. Quantitative HPLC
analysis of acidic opines by phenylthiocarbamyl
derivatization. Anal. Biochem. 174:623-627.
Sato, M., M. Takahara, N. Kanno, Y. Sato, and W. R.
Ellington. 1987. Isolation of a new opine, 0-alanopine, from the extracts of the muscle of the
marine mollusc, Scapharca broughtonii. Comp.
Biochem. Physiol. 88B:803-806.
Seals, J. D. and S. H. Grossman. 1988. Purification
and characterization of arginine kinase from the
sea cucumber Caudina arenicola. Comp. Biochem. Physiol. 89B:701-707.
Shick, J. M., E. Gnaiger, J. Widdows, B. L. Bayne, and
A. de Zwaan. 1986. Activity and metabolism in
the mussel Mytilus edulis L. during intertidal hypoxia and aerobic recovery. Physiol. Zool. 59:627642.
Shick, J. M., J. Widdows, and E. Gnaiger. 1988.
Calorimetric studies of behavior, metabolism and
energetics of sessile intertidal animals. Amer. Zool.
28:161-181.
Siegmund, B., M. Grieshaber, M. Reitze, and E. Zebe.
1985. Alanopine and strombine are end products
of anaerobic glycolysis in the lugworm, Arenicola
marina L. (Annelida, Polychaeta). Comp. Biochem. Physiol. 82B:337-345.
Simon, L. M., J. Theodore, and E. D. Robin. 1978.
Regulation of biosynthesis/biodegradation of oxygen-related enzymes by molecular oxygen. In E.
D. Robin (ed.), Extrapulmonary manifestations of
respiratory disease, pp. 151-169. Marcel Decker,
Inc., New York.
Somero, G. N. 1983. Environmental adaptation of
proteins: Strategies for the conservation of critical
functional and structural traits. Comp. Biochem.
Physiol. 76A:621-633.
Somero, G. N. and J. J. Childress. 1980. A violation
of the metabolism-size scaling paradigm; activities
of glycolytic enzymes in muscle increase in largersize fish. Physiol. Zool. 53:322-337.
Spicer, J. I., A. C. Taylor, and A. D. Hill 1988. Acidbase status in the sea urchins Psammechinus miliaris and £c/j/m«escM/e«/u5(Echinodermata: Echinoidea) during emersion. Mar. Biol. 99:527-534.
Storey, K. B. 1986. Aspartate activation of pyruvate
kinase in anoxia tolerant molluscs. Comp. Biochem. Physiol. 83B:807-812.
Towe, K. M. 1981. Biochemical keys to the emergence of complex life. In J. Billingham (ed.), Life
534
DAVID ROBERT LIVINGSTONE
in the universe, pp. 297-306. The MIT Press, Cambridge, Massachusetts.
Van Aardt, W. J. and C. T. Wolmarans. 1987. Effects
of anoxia on the haemolymph physiology and lactate concentrations in the freshwater crab Potamon warreni Caiman. Comp. Biochem. Physiol.
88A:671-765.
Volckaert, F. and E. Zouros. 1989. Allozyme and
physiological variation in the scallop Placopecten
magellanicus and a general model for the effects
of heterozygosity on fitness in marine molluscs.
Mar. Biol. 103:51-61.
Widdows, J., R. I. E. Newell, and R. Mann. 1989.
Effects of hypoxia and anoxia on survival, energy
metabolism, and feeding of oyster larvae (Crassostrea virginica, Gmelin). Biol. Bull. 177:154-166.
Zange, J., H.-O. Portner, and M. K. Grieshaber. 1989.
The anaerobic energy metabolism in the anterior
byssus retractor muscle of Mytilus edulis during
contraction and catch. J. Comp. Physiol. 159B:
349-358.