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Plant Cell Physiol. 39(5): 467-473 (1998)
JSPP © 1998
Mini Review
Revision of Biochemical pH-Stat: Involvement of Alternative Pathway
Metabolisms
Katsuhiro Sakano
National Institute of Agrobiological Resources, Department of Plant Physiology, Tsukuba, Ibaraki, 305 Japan
A new hypothesis is proposed to explain the metabolic
regulation of intracellular pH (especially of the cytoplasm)
in plants and replace the classic biochemical pH-stat
[Davies (1973) Symp. Soc. Exp. Biol. 27: 513]. Alternative
route glycolysis [Plaxton (1996) Annu. Rev. Plant Physiol.
Plant Mol. Biol. 47: 185], alternative pathway respiration
[Day et al. (1996) Plant Physiol. 110: 1], and malate-derived lactic and alcoholic fermentation (alternative pathway fermentation), all unique to plants, are organized into
a novel mechanism that functions in toto as biochemical
pH-stat. Its uniqueness to plants is examined from the
evolutionary viewpoint that plants adopted the proton as
the ion with which to create the electrochemical potential
difference across the plasma membrane to energize uphill
transports ("proton system"). The biochemical pH-stat is
assumed to have evolved as a security mechanism for the
"proton system" so that it could cope with the potential acidification of the cytoplasm under environmental stress.
Key words: Alternative pathway fermentation — Alternative pathway glycolysis — Alternative pathway respiration —
Biochemical pH-stat — pH regulation — Proton system.
Regulation of intracellular pH, especially of the cytoplasm is one of the most basic homeostatic processes on
which metabolism, transport, signaling, etc. in living organisms depend. In plant cells, two mechanisms have been considered to be responsible for this regulation (Smith and
Raven 1979, Kurkdjian and Guern 1989). One of them is a
biophysical mechanism in the plasma membrane: an electrogenic, ATP-driven proton pump that extrudes intracellular protons out of the cell against the pH gradient and
electrostatic force across the membrane. Another is a biochemical mechanism in the cytoplasm: pH-sensitive carboxylation and decarboxylation reactions in organic acid metabolism that produce and consume protons (Davies 1973,
1986).
In the present paper, I discuss the metabolic regulation
of the cytoplasmic pH in plant cells and propose a new hypothesis. Little attention will be paid to the ion transport
systems at the plasma membrane and the tonoplast, though
they are known to play basic roles in the intracellular pH
regulation (see, Smith and Raven 1979, Kurkdjian and
Guern 1989). Rather, the main focus will be on the metabolic regulation of the cytoplasmic pH under conditions in
which the capacity of such biophysical mechanisms are insufficient to cope with the "pH load", or the mechanisms
are inhibited by various environmental stresses.
Proton System and its Security Mechanism
Sodium system and proton system—Intracellular pH
regulatory mechanisms such as the proton pump and organic acid-based biochemical pH-stat seem to be common and
unique (if not exclusive) to the plant kingdom. This commonality and uniqueness point to the primary significance
of proton in plants. During evolution, animals incorporated the "ancient ocean" inside their bodies as the interstitial environment (body fluid) rich in NaCl (Baldwin
1949). Therefore, it is no wonder that they adopted (or
sustained) the ATP-driven N a + / K + pump as the primary
pump at the interface of the cell and its surroundings (i.e.,
plasma membrane), creating an electrochemical potential
gradient of sodium to energize uphill transports across the
plasma membrane ("sodium system"). The major intracellular pH regulation in animals, thus, depends on secondary
transport of protons utilizing the electrochemical potential
gradient of sodium (i.e., sodium/proton antiport) (Madshus 1988).
In contrast, plants which are considered to have evolved from freshwater green algae (Graham 1985, Hori et al.
1985), abandoned the sodium system and employed, instead, the "proton system", in which electrochemical potential differences of protons across the plasma membrane
were employed to energize secondary transport'. This was
probably because their environments, freshwater and land,
were no longer rich in sodium (or other cations to replace
1
Although sodium-dependent inorganic phosphate transport is
known in many freshwater alga, sodium is not an essential
nutrient for eukaryotic freshwater algae or higher plants (Raven
and Smith 1980).
Abbreviations: F1.6BP, fructose-1,6-biphosphate; F6P, fructoses-phosphate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; PFK, phosphofructokinase.
467
468
Revision of biochemical pH-stat
it). This proton system acquired during evolution was a
wise selection for plants, which are not mobile. Sodium in
animals must be taken up from the environment, but protons can be produced internally by metabolism.
temative pathway glycolysis
Biochemical pH-stat as security mechanism of the proton system—The strength of the proton system is also its
detriment. The only and critical weakness of the proton system seems to be the acidic environment that cells inevitably
Alternative pathway respiration "|
ICytochrome pathway respiration!
Aerobic proton sink
I H O
Alternative pathway fermentation
Classic Biochemical pH-stat
Fig. 1 The structure and function of the revised biochemical pH-stat in the plant cell. (Structure) Heavy and dotted arrows indicate the
main flow of the metabolism related to the function of the pH-stat and its regulation (activation: © and inhibition: ©) by metabolites, respectively. Red arrows with H + point to the proton-consuming reactions, whereas (2e~ + H + ) indicates electron-plus-proton equivalent
of NADH. The pH-stat consists of four functional units of metabolism. The left block (from glucose to pyruvate via malate, on green
and purple plates) denotes the alternative glycolytic pathway, which functions as a proton source (green plate) and pH-sensitive trigger
of the pH-stat (purple plate). The protonogenic reactions are: hexokinase ® , phosphofructokinase (PFK) © , and glyceraldehyde phosphate dehydrogenase ® . The pH-sensitive trigger unit of the pH-stat is essentially the same as that in the classic biochemical pH stat
(Davies 1986) except that the pyruvate kinase ® reaction (on gray plate) is drawn isolated from the main route. The upper right block
(alternative pathway respiration ® , on pink plate and cytochrome pathway respiration @, on yellow plate) and the lower right block
(alternative pathway fermentation, on blue plate) are the proton sink units of the pH-stat under aerobic and anaerobic conditions, respectively. (Function) In response to alkaline-pH stimulus, activity of PEP carboxylase ® increases according to the pH-activity curve
shown in the inset, resulting in promotion of the carbonic anhydrase reaction ® (protonogenic) and activation (de-inhibition) of PFK ©
due to consumption of PEP and production of P; by PEP carboxylase (see Plaxton 1996). OAA (oxaloacetate) is reduced to malate by
malate dehydrogenase ® (proton-consuming). By the conversion of 1 mol of glucose to 2 mol of malate, 4 eq of protons are produced.
Upon acid-pH stimulus, activity of malic enzyme © increases according to the pH-activity curve (inset) and produces more pyruvate,
NADH, and CO 2 , of which, under aerobic conditions, pyruvate (Millar et al. 1996, Hoefnagel et al. 1997) and NADH (Umbach and
Siedow 1993) activate the alternative pathway respiration ® and CO 2 inhibits the cytochrome pathway respiration ® (Gonzalez-Meier et
al. 1996), resulting in diversion of more electrons (and hence, more protons) to the alternative pathway. Although the cytochrome pathway ® is subject to control by energy charge (AE), the alternative pathway ® is not. Thus, any excess protons that acidify the cytoplasm
and activate malic enzyme are destined to quick disposal preferentially through the alternative pathway respiration irrespective of the
energy charge of the cell. Under anaerobic conditions Gower right block), pyruvate and NADH, derived from the malic enzyme reaction,
are transformed either to lactate by lactate dehydrogenase (fi) or to ethanol by way of pyruvate decarboxylase @ and alcohol dehydrogenase © (Roberts et al. 1992). All of these reactions are proton-consuming. Note that the origin of lactate and ethanol when produced for
pH control is malate, not glucose via pyruvate kinase ® .
Revision of biochemical pH-stat
form around themselves. The electrochemical potential gradient of protons, which is a composite of the pH gradient
and membrane potential difference across the plasma membrane generated by the electrogenic proton pump, serves
as the driving force of secondary active transport, such
as proton/substrate symports and proton/substrate antiports. Since these secondary active transports carry protons into the cytoplasm, the potential hazard of cytoplasmic acidification always exists unless the proton pump at
the plasma membrane extrudes them at a comparable rate.
Indeed, cytoplasmic acidification has been shown under
environmental stresses that affect proton pumping (for
review, see Kurkdjian and Guern 1989), and even during
normal physiological processes that cause excess proton influx over efflux (Sakano et al. 1992). This seems to be the
primary reason why the plant cell has evolved a unique
"biochemical pH-stat" as a security mechanism for the proton system.
Classic " biochemical pH-stat" hypothesis—The classic
"biochemical pH-stat" hypothesis was proposed as a fine
tuning mechanism for cytoplasmic pH (Davies 1973, 1986).
The pH-stat consists of a set of carboxylating and decarboxylating enzymes with different pH optima. Of the models
proposed (Davies 1986), the combination of phosphoenolpyruvate (PEP) carboxylase/malic enzyme has been the
most popular and comprehensive. As shown in the inset in
the lower left block of Fig. 1, when the cytoplasmic pH
shifts toward the alkaline range, PEP carboxylase @, having its optimum pH on the alkaline side of the target pH to
which the cytoplasm is to be adjusted, produces more
OAA, which in turn is transformed to malate by malate dehydrogenase © . Since the malate produced is a strong acid,
the pH shift is nullified (Smith and Raven 1979)2. On the
contrary, when the pH shift is toward the acidic range,
malic enzyme © , which has its optimum pH in the acidic
range, would decarboxylate more malate and result in a pH
shift toward the alkaline range. Thus, the cytoplasmic pH
regulation would be established through the synthesis and
degradation of malate by the coordination of the two enzymes.
Why revision of the biochemical pH-stat is required?
—This classic biochemical pH-stat hypothesis (Davies
1973, 1986) together with the biophysical pH-stat hypothesis (Smith and Raven 1979) has been widely accepted, because it explained satisfactorily the changes in endogenous
malate level in response to experimental treatments that
were expected to cause cytoplasmic pH shifts (Haschke and
Liittge 1975, Johnson and Rayle 1976, Stout et al. 1978,
Marre 1979, Romani et al. 1983, Mathieu et al. 1986,
Sakano et al. 1997a, for review, see Smith and Raven 1979,
2
This is not correct. The actual production of H + associated with
malate synthesis is early reactions of glycolysis prior to PEP carboxylase. See next section.
469
Kurkdjian and Guern 1989). However, it has by no means
been proven. Below, I will show two critical shortcomings
of the hypothesis and propose a revision.
Shortcoming 1: Malate synthesis through PEP carboxylase
© (Maruyama et al. 1966) and malate dehydrogenase ©
does not produce but rather consumes a proton near neutral pH.
PEP carboxylase
PEP 3 " + HCO3~ -»• OAA 2 - + P^"
Malate dehydrogenase
-> malate 2 "+NAD +
©
Shortcoming 2: Malate decarboxylation by malic enzyme3
(Macrae 1971) © does not consume a proton.
Malic enzyme
malate 2 - + NAD + - • pyruvate" +NADH + CO2
©
Revision of Biochemical pH-stat
The results of the above examination of the component reactions of the classic biochemical pH-stat are quite
confusing, because they do not seem to play their expected
roles in the pH-stat mechanism. However, if we position
the stat mechanism within the whole flow of the metabolism that precedes and follows it, it becomes clear that they
are playing, though in a limited way, their given roles (see
also Fig. 1).
1. Metabolic flow (glycolysis) preceding the synthesis
of malate through PEP carboxylase and malate dehydrogenase produces protons—When PEP is consumed by PEP
carboxylase © , it is replenished through glycolysis, which
is proton-producing. If the starting material is glucose, the
reactions leading to malate via PEP carboxylase © produce net protons. The protonogenic reactions are:
Hexokinase:
glucose + ATP 4 " -> G6P 2 "+ADP 3 " + H +
Phosphofructokinase:
F6P 2 " +ATP 4 " -> F1.6BP4" + ADP 3 " + H +
Glyceraldehyde phosphate dehydrogenase:
glycelaldehyde-3-P2" + NAD+ + Pj 2 "
-> 1,3-diphosphoglycerate4" + NADH + H +
The rest of the glycolytic reactions leading to PEP neither
consume nor produce a proton. However, if we take
the carbonic anhydrase reaction © (proton-producing)
3
In the original model, Davies (1973) employed the NADP +
malic enzyme (EC 1.1.1.40), but in his revised model (Davies
1986) he suggested the NAD + malic enzyme (EC 1.1.1.39) in the
mitochondria because its response to pH change is much sharper
than that of the NADP + malic enzyme.
470
Revision of biochemical pH-stat
that provides HCO 3 to the PEP carboxylase (Raven and
Newman 1994) and the malate dehydrogenase © reaction
(proton-consuming, as described above) into account, the
balance sheet of protons during metabolic conversion of
glucose to malate is the net production of 2 protons per one
malate produced or 4 protons per one glucose consumed.
Carbonic anhydrase:
CO 2 + H 2 O->HCO 3 ~+H +
©
2. Alternative pathway of glycolysis, the proton
source of the pH-stat—Plaxton (1996) pointed out that the
glycolysis of plants has unique features that are not seen in
other organisms. One is the alternative glycolytic route to
pyruvate that branches at PEP. In addition to the route via
the pyruvate kinase reaction (®, the only path in non-plant
organisms), plants have another route via PEP carboxylase, malate dehydrogenase and malic enzyme reactions.
One of the physiological functions of this route has been
assigned to bypassing the pyruvate kinase reaction during
Pi starvation (Theodorou and Plaxton 1995). Another
unique feature of plant glycolysis is in its mode of control.
In non-plant systems, glycolytic flux is controlled in a
"feed-forward" fashion: activation and de-inhibition of the
first key enzyme PFK © by various effectors (activators:
AMP, fructose-2,6-biphosphate, etc.; inhibitors: ATP,
citrate, etc.) results in an increased level of fructose-1,6-biphosphate (F1,6BP), which in turn activates the second
downstream key enzyme pyruvate kinase ® , so that glycolysis is allowed to proceed. In contrast, in the plant system, the regulation is a feedback process: consumption
of PEP either by pyruvate kinase or by PEP carboxylase
relieves the PEP inhibition of upstream key enzyme PFK.
Moreover, Pj, another product of the PEP carboxylase
reaction, also activates PFK by relieving the PEP inhibition of PFK. From the viewpoint of intracellular pH regulation, feedback is the control mode suitable for avoiding
over-production of protons, because the protonogenic glycolysis is allowed to proceed only when the cytoplasm is so
alkalinized that PEP carboxylase is activated. Such situations may be achieved under aerobic conditions when proton extrusion is active in the presence of external K+
(Sakano et al. 1992, 1997a). In this respect, it is paradoxical
that pyruvate kinase is activated by acid pH under conditions such as anaerobiosis and operation of active transport (Plaxton 1996). This may indicate that, where there is
an urgent requirement of energy, the plant cell dares to accept cytoplasmic acidification that may be compensated
by the operation of the biochemical pH-stat as described
below.
3. Role of PEP carboxylase in the revised biochemical
pH-stat—In the revised pH-stat mechanism, PEP carboxylase © is characterized as the alkaline-pH-sensitive trigger
of the proton generating machinery, glycolysis. Once cyto-
plasmic pH shifts toward the alkaline range, e.g., by proton pumping in the presence of external potassium under
aerobic conditions (Sakano et al. 1997a), the increasing consumption of PEP and simultaneous production of Ps by
PEP-carboxylase open the upstream gate of glycolysis at
PFK © by releasing it from PEP inhibition (Plaxton 1996).
4. Metabolic flow (respiration) following the decarboxylation of malate by malic enzyme consumes protons—Although the malic enzyme reaction © itself does not consume a proton, subsequent respiratory oxidation (®, ®)
of NADH (one of the reaction products of malic enzyme),
and accompanying oxidative phosphorylation ©', if oxidized through cytochrome pathway respiration ®, do consume extra protons.
Respiration:
Oxidative phosphorylation:
ADP 3 " +Pi 2 ~ + H + + energy
ATP 4 " +H 2 O
As will be described below, all the products of the malic enzyme reaction © are feed-forward regulators of the respiratory pathways. Pyruvate is destined to be metabolized in
the mitochondria through the Krebs cycle. But its role is
more than that of a simple substrate of the cycle. The same
is true of the role of NADH in respiration; even CO2 has its
own regulatory function.
5. Alternative pathway respiration, the aerobic proton
sink—Cyanide-resistant alternative pathway respiration is
a common feature of plant cells and tissues (Lance 1991,
Day et al. 1995, Siedow and Umbach 1995, Vanlerberghe
and Mclntosh 1997) and seems as unique as the proton system is to the plant kingdom. Differing from cyanide-sensitive cytochrome pathway respiration, the electron transport from malic enzyme-derived NADH to molecular
oxygen catalyzed by an alternative oxidase is not coupled
to energy conservation (Rustin et al. 1980, Lance et al. 1985)
and, therefore, is free from control by energy charge. This
apparently futile respiration has been shown to increase
under stress conditions: aging (Solomos 1977), wounding,
microbial infection (Uritani and Asahi 1980), low temperature (Purvis and Shewfelt 1993), and treatment with
chemicals (Wagner and Krab 1995) and is also reported to
be involved in thermogenesis (Meeuse 1975) and CAM metabolism (Rustin and Queirotz-Claret 1985, Robinson et al.
1992). Recent, extensive investigations have revealed that
the partitioning of electrons between the cytochrome and alternative pathways is under the regulation of alternative oxidase activity, which is subject to further regulation by a
sulfhydryl-disulfide redox system (Umbach and Siedow
1993) and by allosteric activation by a-keto acids, especially by pyruvate (Millar et al. 1996, Hoefnagel et al. 1997).
Although the "energy overflow" function seems to be the
consensus at this moment (Day et al. 1996, Vanlerberghe
Revision of biochemical pH-stat
and Mclntosh 1997), no convincing general role has been
assigned to alternative pathway respiration that explains its
universal occurrence in plants.
From the foregoing overview, it is now clear that alternative pathway respiration ® , rather than the cytochrome
pathway © , is an integral part of the revised biochemical
pH-stat. Not only serving as an electron donor to the respiratory pathway, NADH would also activate, in feed-forward fashion, the alternative oxidase through the sulfhydryl-disulfide redox system. Moreover, pyruvate, another
reaction product, should also activate the same enzyme in
an allosteric manner. Furthermore, one of the products
of the enzyme reaction, CO 2 , preferentially inhibits cytochrome pathway respiration at low concentration (GonzalezMeier et al. 1996), resulting in closer linking of the malic enzyme reaction with the alternative pathway. Collectively,
all these features of the reaction products of the malic enzyme indicate that, when low pH activates the malic enzyme, the alternative pathway is reserved exclusively for
the oxidation of its products. Independence from energycharge control of the alternative pathway is another
favorable feature of the pH-stat, because the situations
that require pH regulation are usually urgent.
The observation of acid-pH dependent malate oxidation by malic enzyme in plant mitochondria (Macrae 1971,
Tobin et al. 1980), together with its close association to alternative pathway respiration (Rustin et al. 1980), suggests
that malic enzyme and alternative pathway respiration
might respond to acid pH as a pH-stat. Later, this was indeed suggested by Lance and Rustin (1984)4.
Our recent study showed that Pj uptake by Catharanthus roseus cells, which proceeds by the proton cotransport
mechanism (Sakano 1990), acidified the cytoplasm (Sakano
et al. 1992) and activated malate decomposition (Sakano et
al. 1998), was sensitive to inhibition by n-propyl gallate, a
specific inhibitor of alternative oxidase, under conditions
in which cytochrome pathway respiration was inhibited
(Sakano et al. 1997b). Whether alternative pathway respiration can function as a proton sink of the cells for protons
loaded with Pj may be clarified by examining the cytoplasmic pH control in the presence of inhibitors of alternative
oxidase, or, more strictly, by estimating the electron partitioning between the cytochrome and alternative pathways
(Guy et al. 1989, Robinson et al. 1992, Ribas-Carbo et al.
1995).
6. Lactic and alcoholic fermentation, the anaerobic
proton sink—Synthesis of lactate or ethanol has generally
been regarded as the means to regenerate NAD + required
4
To the author's knowledge, this is the first paper that implicated
a connection between the biochemical pH-stat and alternative
pathway respiration. However, this aspect has not been cited in
any major review papers on biochemical pH-stat or on alternative
pathway respiration so far published, although it was briefly
quoted by Lance et al. (1985) themselves.
471
for sustaining glycolytic energy production under anaerobic conditions. In plants, however, cytoplasmic acidification under hypoxic or anoxic conditions has been ascribed
to lactic fermentation (Roberts et al. 1984, but see Vertapetian and Jackson 1997). In the revised biochemical pH-stat,
however, some of the same anaerobic glycolytic reactions
leading to lactate (©) or ethanol (@, ©) from malate-derived pyruvate are regarded as proton sinks as well (lower
right block of Fig. 1). As pointed out by Hochachka and
Mommsen (1983), lactate formation from glucose through
glycolysis via the pyruvate kinase reaction ® produces one
proton per one lactate formed, and ethanol formation
from glucose via the same reaction neither produces nor
consumes a proton per one ethanol formed 5 . However, if
they are formed from malate via the malic enzyme reaction
© , more protons are consumed (one proton per one lactate
and two protons per one ethanol, see Fig. 1).
Stimulation of malic enzyme (Edwards and Roberts
1996) and formation of lactate and ethanol from malate
(Roberts et al. 1992) have been demonstrated in corn root
tips under anaerobic conditions that acidified the cytoplasm. In Catharanthus cells, anoxia-induced accumulation of lactate has been suggested to derive from malate
(Sakano et al. 1997a). The induction of lactate dehydrogenase by hypoxic treatment of plant root tissues (Hoffman et
al. 1986, Hondred and Hanson 1990) is apparently un-physiological if it is to synthesize more lactate from glucose that
should intensify the cytoplasmic acidification (Ratcliffe
1995). However, if the induction is for lactate synthesis
from malate, it should ameliorate the cytoplasmic acidification under hypoxia. Ethanol accumulation upon treatment
with antimycin A (inhibitor of cytochrome pathway) in the
transgenic tobacco leaf that lacked alternative oxidase
(Vanlerberghe et al. 1995) is consistent with the proposed
pH-stat mechanism under anaerobic conditions, but further experimental evidence is needed to verify this hypothesis.
Lactate dehydrogenase:
pyruvate ~ + NADH + H+ -> lactate ~ + NAD+
Pyruvate decarboxylase:
pyruvate~ + H + -* acetaldehyde + CO2
Alcohol dehydrogenase:
acetaldehyde + NADH+ H + -+ ethanol + NAD +
(Q)
@
<Q>
7. Role of malic enzyme in the revised biochemical
pH-stat—Malic enzyme © is characterized as the acid-pHsensitive trigger of the proton consuming machinery: the alternative pathway respiration (under aerobic conditions)
5
Stoichiometry if synthesized ATP is used (i.e., hydrolyzed to
produce energy)
l/2Glucose -»• Lactate" + H + + energy
l/2Glucose-> Ethanol+CO 2 +energy
Revision of biochemical pH-stat
472
and lactic and/or alcoholic fermentation (under anaerobic
conditions).
Concluding Remarks
Dehydrogenation is a common way of biological oxidation. The reducing equivalent (2H), taken from substrates
in the form of NAD(P)H + H + , is finally oxidized (through
respiration) by molecular oxygen to form water. We should
remember that one side of respiration is the electron transport to oxygen, but the other side is the protonation of the
"electronized" oxygen to form water, i.e., proton consumption. Alternative pathway respiration is common and
unique, if not exclusive, to the plant kingdom. Its protonconsuming function, which is not only free from regulation
by energy charge but is also subject to activation by the
reaction products of malic enzyme in a feed-forward manner, strongly suggests that the main function of alternative
pathway respiration is the pH regulation of the cytoplasm.
This well explains the universal occurrence of this respiration among the plant kingdom, behaving as an integral part
of the security mechanism of the "proton system" adopted during evolution. The absolute requirement of a-keto
acids, especially pyruvate, for AOX activation (Millar et al.
1996, Hoefnagel et al. 1997) strongly supports the hypothesis. Other physiological functions, such as CAM, thermogenesis, and avoidance of active oxygen formation, so
far implicated in this respiration (for review, see Lambers
1985, Wagner and Krab 1995) may be the evolutionary
variations of this unique respiration adapted to various
environmental stresses in which rapid consumption of
electrons and/or protons are urgent or profitable to the
plants. Although its involvement in eliminating excess
photosynthetic reducing power through photorespiration
(Vanlerberghe and Mclntosh 1997) has been suggested, no
conclusive evidence has been presented.
In addition, it should be noted that the apparently
futile conversion of PEP via malate to pyruvate and further to lactate or ethanol without conserving energy6 in the
plant glycolytic route may be called, by analogy to alternative pathway respiration, "alternative pathway glycolysis"
and "alternative pathway fermentation", respectively. This
is because their common physiological function is "pH regulation at the expense of otherwise possible energy conservation". In other words, pH regulation through the alternative pathways is not free of cost.
An alternative oxidase similar to the plant enzyme has
been found in the protozoa trypanosome (Clarkson et al.
1989). However, its function does not seem to involve pH
regulation. The protozoan alternative oxidase is linked
physically in the mitochondrial membrane to glycerol-36
Synthesis of lactate or ethanol from PEP via pyruvate kinase
produces one ATP.
phosphate dehydrogenase (@, not shown in Fig. 1); the
reaction catalyzed is protonogenic:
sn-glycerol-3-phosphate2~+NAD+ ->
dihydroxy acetone phosphate 2 " + NADH + H +
®
Subsequent oxidation of the product, NADH, by the alternative oxidase is accompanied by the consumption of the
proton produced simultaneously in the dehydrogenase reaction © itself and, therefore, would not consume a proton
produced (or emerging) elsewhere in the cell (acidifying the
cytoplasm).
Thanks to the development of DNA-data bases, more
"alternative oxidases" will be discovered in the future in the
diverse range of organisms outside of the plant kingdom.
The author shares the thought that "There are more things
in heaven and earth, Horatio, than are dreamt of in your
philosophy" (Shakespeare, from The Tragedy of Hamlet,
Prince of Denmark) with those working in the field of alternative pathway respiration (Day et al. 1996).
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Revision of biochemical pH-stat
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(Received September 11, 1997; Accepted March 30, 1998)