Download Facultative Anaerobiosis in the Invertebrates: Pathways and Control

A M . ZOOLOGIST, 11:125-135 (1971)
Facultative Anaerobiosis in the Invertebrates:
Pathways and Control Systems
HOWARD J. SAZ
Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556
SYNOPSIS. An increasing number of the invertebrates studied have been found to rely
on an anaerobic energy metabolism during at least one stage in their development.
Some of the bivalve mollusks, and particularly a relatively large number o£ parasitic
nematodes, cestodes, trematodes and acanthocephala may be classified in this category.
Regardless of the aerobic or anaerobic requirements of the parasitic helminths, two
features appear to be common to all members of this group which have been
examined. First, they are all capable of taking up oxygen. Second, none can oxidize
substrates completely to CO2 and H2O; end-products of fermentation invariably
accumulate, indicating either the complete absence or the presence of only a limited
terminal respiratory pathway.
The intestinal nematode, Ascaris lumbricoides, has served as a model system for the
elucidation of an anaerobic energy-yielding pathway of carbohydrate dissimilation
which appears to be operative in the anaerobic stages of a number of other invertebrates. This pathway differs in several major respects from those previously described
for mammalian and other aerobic tissues. The Ascaris system is discussed in detail and
is compared with other invertebrate and vertebrate metabolisms.
For many years it was thought that essentially all multicellular tissues obtained
their energy by employing similar aerobic
terminal respiratory pathways. With the
advent of biochemical investigations of
some of the invertebrate groups, however,
it became apparent that not all tissues had
an aerobic requirement to fulfill their energy needs. It was suggested by von Brand
(1950), Bueding (1949) and others since,
(Saz, 1969), that the adult forms of the
larger intestinal helminths, for example,
possess a predominantly anaerobic energy
metabolism. The early suggestions were
based primarily on the fact that these
worms reside in the intestine, an environment where the oxygen tension is very
low. Further experimentation has substantiated these predictions for an increasing
number of helminth parasites.
It has been suggested also, by the reports
of Galtsoff (1964) as well as those of
Hammen and collaborators (See Wegener
et al., 1969), that some of the bivalve mollusks, particularly the American oyster,
tolerate long periods of anaerobiosis by
utilizing energy-producing pathways simiThe author's current investigations are supported in part by the NIH, U.S. Public Health Service
(Grant AI-09483).
lar to those which occur in the intestinal
nematode Ascaris lumbricoides. It appears
likely that other invertebrates which depend upon intermittent or continuous
periods of anaerobiosis may employ similar
metabolic pathways.
It would seem a simple matter to determine whether or not an organism requires
oxygen for survival. In the case of many
helminths, however, this basic question has
remained unanswered for a number of
reasons. All nematodes, as well as cestodes and trematodes which have been examined thus far, are capable of taking up
oxygen when incubated in vitro in air.
This includes Ascaris. On the other hand,
the physiological significance of this oxygen uptake has been seriously questioned
in the case of those worms which normally
dwell in essentially anaerobic environments, such as the lumen of the intestinal
tract. In addition, some of the intestinal
helminths have been shown by a number
of investigators to survive for extended
periods of time in anaerobic environments.
Oxygen may even be detrimental to survival in some instances. More recently,
Berntzen (1961) and subsequently Schiller
(1965) have cultured successfully the rat
tapeworm, Hymenolepis diminuta. In
125
126
HOWARD J. SAZ
agreement with earlier biochemical findings, the worms were cultured under
anaerobic conditions, oxygen being detrimental to development. It would appear,
therefore, that at least some of the helminths are anaerobes in the adult stage. It
is particularly intriguing that none of the
helminths studied to date, regardless of
their oxygen requirements, be they aerobic
or anaerobic, are capable of the complete
oxidation of substrates to CO2 and water.
All of those examined accumulate organic
end-products. Since substrates are not completely metabolized, it would appear that
terminal respiration is either absent or
rate-limiting in this entire group of organisms.
Although fermentation products always
accumulate, they differ both qualitatively
and quantitatively with each parasite. It is
of interest, however, that a surprising
number of parasitic helminths accumulate
one product in common. That is succinate.
Representative nematodes which form
succinate or products presumably derived
from succinate include Ascaris lurnbricoides, Heterakis gallinae, Trichuris vulpis, and the larval form of Trichinclla spirails. Cestodes which also show this property include the tapeworms Hymenolepis diminuta and Moniezia expansa, and cysts of
Echinococcus granulosus and Taeiiia
taeniaformis. The liver fluke, Fasciola hepatica and Moniliformis dubins represent
trematodes and acanthocephalans, respectively, which fall into the category of succinate accumulating helminths (For references see Sa/. and Bueding, 1966).
The invertebrate which has served as a
model system for biochemical studies of
this type has been the intestinal parasite,
Ascaris lumbricoides. To a large extent,
the anaerobic pathway for carbohydrate
dissimilation has been elucidated in this
nematode, and may be related to pathways
occurring in other helminths and possibly
in other groups of facultative invertebrates.
In spite of the fact that Ascaris adults
consume oxygen in vitro (Laser, 1944), as
do all other helminths examined, early
studies indicated that this parasite derived
its energy from an anaerobic sequence of
reactions. Weinland (1901) reported that
Ascaris survives equally well under aerobic
or anaerobic conditions, indicating that
the adult stage obtains its energy via
anaerobic pathways of metabolism. In accord with this, the organism is not sensitive to cyanide, and neither cytochrome c
nor cytochrome oxidase is demonstrable in
Ascaris tissues (Bueding and Charms,
1952). It would appear that a flavin system
for terminal oxidation has replaced the
cytochrome system, since, in the presence
of air, hydrogen peroxide is formed
(Laser, 1944). Hydrogen peroxide is toxic
to the worm due to a deficiency of catalase
(Laser, 1944; Magath, 1918). A further
indication of a flavin system is the finding
that the rate of oxygen utilization is dependent upon the oxygen tension, being
almost negligible at the oxygen tensions
found in the physiological environment of
the adult ascarids (Bueding and Charms,
1952; Harnisch, 1933; Laser, 1944; Rathbone, 1955). One additional indication
that the parasite is an anaerobe is that,
qualitatively at least, the same products are
formed in either air or N2-CO2 environments (Harpur and Waters, I960). These
observations are consistent with the conclusion that this parasite is predominantly
anaerobic and, unlike mammalian tissues,
possesses a terminal flavin, rather than a
cytochrome oxidase system.
Succinate and a mixture of volatile fatty
acids comprise the major end-products of
Ascaris fermentations. With the aid of
C u labeled substrates, Saz and Vidrine
(1959) studied the anaerobic pathway for
succinate formation in Ascaris and suggested the pathway outlined in Figure 1.
Their findings were in accord with the
dissimilation of glucose via glycolysis to a
three carbon moiety, presumed to be pyruvate. Carbon dioxide was then fixed into
the three carbon compound with subsequent reduction of the product to succinate. Succinate and pyruvate were then
found to be precursors of the volatile fatty
acid end-products. Of particular interest
was the suggestion that succinate was
formed by a reversal of the reactions which
ANAEROBIOSIS IN INVERTEBRATES
FIG. 1. Proposed pathway for the formation of
succinate and volatile acids in Ascaris muscle.
occur in aerobic tissues, that is, by the
backward or reductive reactions of the tricarboxylic acid cycle. Succinate dehydrogenase in the parasite muscle acted physiologically in a manner opposite to that of
mammalian tissues. Rather than a succinate dehydrogenase, Ascaris appeared to
possess a fumarate reductase which would
serve to reoxidize the reduced DPN
formed during glycolysis. Kmetec and
Bueding (1961) partially purified Ascaris
succinate dehydrogenase and demonstrated
that it did indeed behave as a fumarate
reductase system. Studies of this nature,
together with the report of Seidman and
Entner (1961), made it clear that Ascaris
muscle relied on this backward pathway
for much of its energy supply, since ATP
generation was found to be associated with
the electron transport system coupled to
the fumarate reductase reaction. Fumarate
acts as the ultimate electron acceptor,
reoxidizing reduced DPN with the concomitant formation of succinate. The transfer of electrons from DPNH to an intermediate flavin presumably results in the liberation of energy in the form of ATP. In
Ascaris, therefore, fumarate takes the place
of oxygen as the terminal electron acceptor.
What, then, are the controlling factors
which allow this parasite to maintain its
metabolic flow in a direction opposite to
that found in most other tissues? A hint at
an answer to this question came with the
demonstration of the presence of an active phosphoenolpyruvate carboxykinase in
Ascaris muscle (Saz and Lescure, 1967). It
127
can be noticed that the accumulation of
succinate by Ascaris requires the fixation
of one molecule of COL. for each succinate formed. Phosphoenolpyruvate carboxykinase is an enzyme which has CO2
fixing capabilities and which was first
described in chicken and, subsequently,
mammalian liver by Utter and Kurahashi
(1954). In contrast to Ascaris muscle, the
enzyme could not be detected in mammalian muscle. Prescott and Campbell (1965)
were the first to demonstrate the presence
of this enzyme in a helminth, Hymenolepis
diminuta. Subsequently, it has been
shown to be present in a number of other
helminths. The reaction catalyzed by phosphoenolpyruvate (PEP) carboxykinase is
shown in Figure 2. The enzyme catalyzes
OAA + ITP ?± PEP + CO, + IDP
[GTP]
[GDP]
FIG. 2. Reaction catalyzed by phosphoenolpyruvate carboxykinase.
the reversible decarboxylation of oxalacetate (OAA) in the presence of the nucleotide inosine triphosphate (ITP) to form
phosphoenolpyruvate (PEP), CO2 and
inosine diphosphate (IDP). Guanosine
triphosphate can substitute partially for
the inosine nucleotide. The reverse reaction, the fixation of CO2 into PEP to form
oxalacetate, can also be catalyzed by this
system, but the enzyme in mammalian tissues is thought to catalyze primarily the
decarboxylation of oxalacetate (Utter et
al, 1964). Therefore, once again, Ascaris
appears to catalyze this reaction in a direction opposite to that found in mammalian
tissues. Saz and Lescure (1967) incubated
dialyzed Ascaris muscle homogenates in
the presence of C14 labeled sodium bicarbonate plus unlabeled oxalacetate. After
five minutes of incubation, the incorporation of radioactivity into the OAA was
determined. Results are shown in Table 1.
This assay procedure for PEP carboxykinase indicated a high activity in Ascaris muscle in the presence of ITP.
Guanosine triphosphate (GTP) can partially replace ITP, but activity with ATP is
quite low. Of major importance is the fact
that the reaction is dependent upon the
128
HOWARD J. SAZ
TABLE 1. Incorporation of NaHC^O, into OAA by Asearis Muscle Homogenate (PEP CarboxyTcinase Assay)
System
Dpm fixed/5 min.
/iMoles fixed/5 min.
Complete, with ITP
Complete, with GTP
Complete, with ATP
No nueleotide added
Without Oxalacetate
180,200
125,240
47,000
4,400
7.45
5.18
1.94
0.18
30
addition of a nueleotide. This dependency
for a nueleotide distinguishes the PEP carboxykinase from some of the other known
CO2 fixing enzymes.
These, plus subsequent findings, indicated strongly that PEP carboxykinase is indeed the enzyme responsible for catalyzing
the fixation of CO2 into PEP in Asearis muscle. Presumably the oxalacetate formed by
the reaction ultimately would be reduced
to succinate, thereby making phosphoenolpyruvate rather than pyruvate the
point at which the metabolism of Asearis
muscle branches off from that of the host
tissues.
A new question now presented itself.
Why does the PEP carboxykinase of Asearis act primarily in a direction opposite to
that of the equivalent mammalian enzyme?
The helminth system, on the one hand,
catalyzes the fixation of CO2; the mammalian system, on the other hand, catalyzes
primarily the decarboxylation of oxalacetate, thereby forming PEP, which, in turn,
could be transformed into glycogen by a
reversal of the reactions of glycolysis.
In an effort to determine whether or not
the Ascnris PEP carboxykinase was similar to the mammalian enzyme, assay
procedures were worked out and optimal
conditions determined for assaying the
reaction in both directions (Saz and Lescure, 1969). The worm enzyme, like the
one from the host, was almost twice as
TABLE 2. OpHmal Activities and Apparent
Values of Asearis PEP Carboxykinase
Km
Reaction Assayed
Optimal
Activityz
Apparent Km
for Substrate
M
P E P + CO.. -> OAA
OAA -> P E P + CO2
403
794
2.ti X 10-'3
1.8 X 10-
" Activity expressed
former! /min/mf* protein.
m^mole^ oH product
0
active in the direction of oxalacetate decarboxylation (Table 2). It should be emphasized that these activities are based
upon optimal conditions of substrate concentration. If now the effect of substrate
concentration on enzymatic activity is
studied and the Michaelis constant (Km)
determined, a different picture is obtained.
Knl values designate the concentration of
substrate which results in half maximal
activity. In this respect, it is an approximation of the affinity of the enzyme for the
substrate under investigation. It is of particular interest that with the Asearis enzyme, half maximal activity in the direction of CO2 fixation was reached at approximately one-seventh the substrate concentration of the reverse decarboxylation
reaction (Table 2). These findings indicate that under physiological conditions
the reaction may be catalyzed primarily in
the direction of CO2 fixation. This possiblity becomes particularly impressive in
view of the fact that Asearis tissues contain
surprisingly high concentrations of PEP
which would tend to push the CO2 fixing
reaction. In addition, a very high malate
dehydrogenase activity is also present
which should serve to pull off oxalacetate
rapidly, thereby possibly pulling the enzyme again in the direction of CO2
fixation.
Figure 3 presents a summary of some of
the reactions of Asearis and mammalian
tissues discussed so far. In both parasite
and host tissues, carbohydrate is dissimilated to phosphoenolpyruvate. At this
point, the two types of tissues diverge; Ascaris fixing CO2 via reaction (2), PEP
carbox\kinase, to form oxalacetate which
is ultimately reduced to succinate. Mammalian tissues, on the other hand, generally employ reaction (2) in the opposite
ANAEROBIOSIS IN INVERTEBRATES
FIG. 3. Comparison of metabolic reactions of
mammalian and Ascnri.s tissues. (1) = Pyruvate
kinase. (2) = Phosphoenolypyruvate carboxykinase.
direction. That is, in mammalian tissues,
oxalacetate formed either from succinate
or pyruvate is decarboxylated to PEP which
can then serve as a precursor of glycogen,
the overall process being referred to as
glyconeogenesis. Most significant is the fact
that mammalian tissues utilize PEP rapidly in the presence of ADP to form pyruvate and ATP (reaction 1). This reaction is catalyzed by pyruvate kinase. Once
pyruvate is formed it may be converted
anaerobically (as in rapidly contracting
muscle) to lactate, or aerobically to CO2
and water via the tricarboxylic acid cycle.
The next question which arises is, why is
the enzyme pyruvate kinase so successful
in its competition for substrate PEP in
mammalian tissues, but so unsuccessful in
Ascaris where CO2 fixation appears to be
favored? In an attempt to answer this
question, various enzymatic activities of
Ascaris muscle were determined under optimal conditions (Bueding and Saz, 1968).
PEP carboxykinase was assayed spectrophotometrically in the direction of COo
fixation. Results are presented in Table 3.
The figures in parentheses indicate the
TABLE 3. Enzymatic Activities of Ascaris Muscle
Enzyme
PEP Carboxykinase
Lactate Dehydrogenase
Malate Dehydrogennse
Pyruvate Kinase
Substrate Utilized
(m^moles/min/mg protein)
168 (126-209)
143 (71-163)
5,278 (4,221-6,807)
7 (4.6-9.1)
129
range of activities observed. Lactate dehydiogenase and PEP carboxykinase were
found to be present at comparable activities. This is of interest, in view of the fact
that intact ascarids do not accumulate lactate. As mentioned previously, the malate
dehydrogenase activity is very high, the
highest of all Ascaris enzymes assayed.
Most significant is the barely detectable
activity of pyruvate kinase. This enzyme is
essentially absent from Ascaris muscle. The
very low level of pyruvate kinase activity,
then, explains why pyruvate is not formed
directly from PEP by this parasite. In addition, it would explain why the fixation of
CO2 into PEP occurs as a major metabolic
reaction in this tissue. Since phosphoenolpyruvate is not removed appreciably by
pyruvate kinase in the parasite muscle,
the endogenous levels of PEP and pyruvate
were compared in Ascaris muscle. In accord with predictions, the existing concentrations of PEP were, on the average, approximately twice those of pyruvate (0.26
^moles/gm versus 0.12 ^moles/gm). As
pointed out earlier, this relatively high tissue level of PEP would favor the PEP
carboxykinase reaction in the direction of
CO2 fixation.
Two major questions remained to be answered by the proposed scheme for succinate formation in Ascaris. First, fermentations by this nematode result in the accumulation of relatively large quantities of
acetate. In addition, acetate serves as a
precursor for alpha-methylbutyrate which
is a major fermentation product of the
parasite. If pyruvate can not be formed
from PEP via the pyruvate kinase reaction,
then from where do it and acetate originate in the Ascaris fermentation? Second,
if pyruvate is formed by some pathway in
the muscle, then why does lactate not accumulate in Ascaris fermentations, since
the presence of an active lactate dehydrogenase has been demonstrated? Recent findings have helped elucidate answers to both
questions.
Saz and Hubbard (1957) described an
active DPN linked malic enzyme from Ascaris muscle. This enzyme catalyzes the
reaction shown in Figure 4. Malate is ox-
130
HOWARD J. SAZ
1-Malate + DPN ^ Pyruvate + CO2 + DPNH + H+
FIG. 4. Reaction catalyzed by the malic enzyme.
idatively decarboxylated in the presence of
DPN to pyruvate, CO2 and DPNH.
Although this reaction is reversible, the
Ascaris system is approximately 300 times
faster in the direction of pyruvate formation from malate. This reaction then could
serve as a means of pyruvate formation in
Ascaris muscle, which would be independent of pyruvate kinase. Despite the fact
that the malic enzyme was found in Ascaris
muscle in 1957, its physiological function
could not be understood until quite recently.
Although the malic enzyme could
provide a means for forming pyruvate, the
question concerning the lack of lactate
formation remained to be answered. One
possible answer was compartmentalization
within the cell such that pyruvate and lactate dehydrogenase would not appear
within the same compartment.
Ascaris has adapted itself remarkably
well to its environment. Mitochondria are
present in Ascaris tissues, but they function
in a manner quite different from the corresponding organelles of the host tissues.
That is, they function anaerobically. The
distribution of several enzyme systems from
Ascaris muscle between the soluble and
mitochondrial fractions was determined by
Saz and Lescure (1969). The enzymes examined were malic enzyme, fumarase, PEP
carboxykinase and lactate dehydrogenase
(Table 4). Ascaris muscle was homogenized and large particles removed by slow
speed centrifugation. The 128g supernatant obtained contains both the mitochondria and the soluble fraction. This fraction
contains essentially all of the activities
being examined. If now we sediment the
mitochondria by centrifugation at 9,000g
the supernatant contains the so-called soluble fraction. It should be noted that all of
the lactate dehydrogenase is recovered in
this soluble fraction; none is retained by
the mitochondria. Similarly, essentially all
of the PEP carboxykinase is recovered in
this fraction, plus a small amount in the
wash of the mitochrondria. In the case of
fumarase, approximately one-half was
recovered in the soluble fraction; the remainder stayed with the mitochondrial
pellet. In the case of the malic enzyme,
approximately one-sixth of the activity remained in the supernatant; the remainder
appeared in the mitochondrial fraction.
YVhen the mitochondria were disrupted,
most of the remaining activities of both
malic enzyme and fumarase were liberated.
Of interest now, if the disrupted mitochondrial fraction was spun at 96,000g
essentially all of the malic enzyme activity
was solubilized, whereas three-fourths of
the fumarase activity remained in the insoluble (membrane-bound) fraction. The
finding, therefore, of small quantities of
malic enzyme activity in the 128g supernatant fraction may reflect a partial disruption of the mitochondria during preparation followed by leakage of the enzyme
TABLE 4. Cellular Distribution of Ascaris Muscle Enzt/mrs
TJmtsVgm of Muscle
Fraction
128<7 Supernatant
9,0000 Supernatant
Mitochondrial Wash
Disrupted Mitochondria
9f>,000<7 Supernatant of Disrupted
Mitochondria
96,0000 Residue of Disrupted
Mitochondria
Malic Enzyme
Fumarase
6.13
1.37
0.70
3.90
3.6S
11.32
6.47
0.03
4.52
1.35
—
3.24
• Units express ^moleh of substrate utilized per minute.
PEP Carboxykinase
Lactate Dehydrogenase
7.60
7.06
0.38
2.05
2.02
0
II
—
0
0
n
—
ANAEROBIOSIS IN INVERTEBRATES
T.
FIG. 5. Pathway of carbohydrate dissimilation in
Ascaris muscle.
into the soluble fraction.
Dissimilatory pathways in mammalian
tissues may now be compared with our
current concepts of the comparable situation in Ascaris. The overall pathway by
which mammalian tissues dissimilate carbohydrate is briefly described as follows.
In the soluble, or cytoplasmic, portion of
the cell, glucose arising either externally or
from glycogen breakdown is phosphorylated and transformed by the glycolytic enzymes into two moieties of three carbons
each, which are converted ultimately to
phosphoenolpyruvate.
Phosphoenolpyruvate, in turn, donates its high energy
phosphate bond to ADP to form ATP and
pyruvate, the reaction being catalyzed by
pyruvate kinase. In mammalian tissues, pyruvate may then be converted to lactate, or
it may enter the mitochondrion where it
undergoes complete oxidation to CO2 and
water via the aerobic reactions of the tricarboxylic acid cycle. These reactions are
coupled through the cytochrome electron
transport system to oxygen.
In view of the more recent findings with
Ascaris, the situation in the parasite muscle may be summarized as illustrated in
Figure 5. According to the proposed pathway, the glycolytic enzymes of Ascaris,
which are present in the soluble portion of
the cell, function similarly to those of the
host tissues up to the point of PEP accumulation. Since phosphoenolpyruvate
can not be dephosphorylated directly to
pyruvate, the Ascaris PEP carboxykinase,
which is found in the soluble portion of
the cell, fixes COS into this compound,
131
resulting in the formation of oxalacetate.
This dicarboxylic keto acid is rapidly reduced by glycolytically formed DPNH, the
reaction being catalyzed by the very active
cytoplasmic malate dehydrogenase. It is
this reaction which serves to regenerate cytoplasmic DPN, a function carried out by
lactate dehydrogenase in mammalian tissues.
Malate thus formed in the cytoplasm
now crosses over into the mitochondrion
and becomes the mitochondrial substrate.
Since direct oxidative systems are absent in
the Ascaris mitochondrion, malate must
be utilized further by a dismutation system. Intramitochondrial reducing power,
in the form of DPNH, is generated by the
oxidative decarboxylation of malate to pyruvate and CO2, thereby giving rise to pyruvate in the absence of pyruvate kinase.
This reaction is, of course, catalyzed by the
mitochondrial malic enzyme. DPNH
formed from this reaction then serves to
reduce a corresponding amount of malate
to succinate via fumarate and the fumarate
reductase reaction with the concomitant
formation of ATP. Pyruvate may then
serve as a precursor for acetate, but again
within the mitochondrion. This reaction
would presumably result in additional reducing power inside the mitochondrion
which might enter into the reductive
formation of the volatile fatty acids which
accumulate during Ascaris fermentations.
The site of fatty acid formation is, however, still not known. It should be recalled
that pyruvate, according to this scheme, is
formed within the mitochondrion, while
lactate dehydrogenase is a cytoplasmic enzyme. Therefore, lactate would not be an
end-product of Ascaris fermentations by
intact worms. It might be expected, however, that homogenates of Ascaris muscle in
which the mitochondria are disrupted and
the lactate dehydrogenase released would
accumulate lactate. This is indeed the
case. Homogenates of Ascaris muscle do
form lactate.
According to this proposed scheme,
malate should be the mitochondrial substrate which would dismutate to a mixture
of pyruvate, succinate and possibly other
132
HOWARD J. SAZ
TABLE 5. The Anaerobic Utilization of Malate by
Intact Asearis Muscle Mitochondria
Compound
Recovered
(jiMoles)
Acetate
Propionate
Pyruvate
Succinate
2.05
0.97
5.25
5.29
Malate Disappearance = 13.29 ^Moles.
volatile acids. In addition, since evidence
has been presented indicating an electrontransport associated phosphorylation resulting from the fumaric reductase reaction,
an uptake of inorganic P32 into organic
phosphate would be expected. Incubation
of L-malate with intact Asearis mitochondria under anaerobic conditions (Saz and
Lescure, 1969) results in the accumulation
of acetate, propionate, pyruvate and succinate (Table 5). These are the products
which might be predicted, and essentially
one pyruvate accumulates for each succinate formed. Presumably the acetate and
propionate arise from pyruvate and succinate respectively.
Asearis mitochondria also catalyze a rapid anaerobic malate dependent phosphorylation as determined by the incorporation
of inorganic P32 into organic phosphates
Mitochondria were again incubated under
a nitrogen atmosphere. In the absence of
malate, less than 0.1 //.moles of P32 are
incorporated. In the presence of malate
and P32, 11.6 ^moles of malate disappeared and 5.4 ^moles of P32 were esterifled in 20 minutes. In place of a P/O
ratio, we may calculate a P32/malate ratio which, in this experiment, was 0.47.
Since, for each mole of malate used, only
one-half goes to succinate, the theoretical
ratio would be 0.5. Therefore, the biochemical behavior of Asearis mitochondria
is in direct accord with the proposed pathway.
At this point, it might be wise to consider whether the anaerobic pathways discussed are peculiar or unique to Asearis.
Apparently this is not the case. Although
information is still incomplete, evidence is
accumulating that more and more of the
anaerobic or facultative anaerobic inverte-
brates may obtain energy by similar
mechanisms. Fairbairn et al., (1961) have
shown that the rat tapeworm, Hymenolepis diminuta, forms succinate and requires
CO2 for glycolysis to proceed. More recently, evidence has been presented indicating a pathway in H. diminuta similar to
that of Asearis (Scheibel and Saz, 1966). A
CO2 requirement has been demonstrated
for a number of other helminths. Ward et
al., (1968rt,fr) have demonstrated an almost
complete lack of pyruvate kinase in larvae
of the nematode Haemonchus contortus as
well as the presence of PEP carboxykinase
and a malic enzyme. Similarly, Prichard
and Schofield (1968a,b) reported low pyruvate kinase and high PEP carboxykinase
activities in the adult liver fluke, Fasciola
hepatica. The findings of Wegener et al.,
(1969) that the American oyster during
anaerobic incubation accumulates succinate, but no lactate, and possesses an active fumaric reductase are particularly intriguing.
Considerable progress has been made in
the area of the biochemistry of invertebrates over the past ten years. However,
with the possible exception of the insects,
comparatively little is known of this vast
group of organisms. There is still much
room for more progress in the years to
come, particularly if one can envision the
large gaps in our knowledge of invertebrate biochemistry, and can discard the
concept that these organisms are necessarily
biochemical miniatures of all other tissues.
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ANAEROBIOSIS IN INVERTEBRATES
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