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OSCILLATORY PHENOMENA IN BIOCHEMISTRr 752

BENNO HESS AND ARNOLD BOITEUX
Max-Planck-Institut fur Ernahrungsphysiologie, Dortmund, West Germany

CONTENTS
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INTRODUCTION. . ..... ............. . .......... ...... .... .. ... ... ....... 237
SOLUBLE OSCILLATORS.. ...... ....... . . . ..... .... . .. .. .. .. .. ...... ..... 239
Enzymes. . . . ... .......... ........ . . ................... ... .........
. 239
Glycolysis in yeast extracts. . . . . . . ... ... ....... .. .. . . ........ .. .... . ... 240
Oscillating glycolys1,s in other systems. . .... . ....... . ............. . .. . ..
. 243
MITOCHONDRIAL SYSTEMS.. ........................... . . ............... 243
CELLULAR SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 247
Metabolic oscillations. . .. ... . ... . . . ... . ....... . ................... . ... 247
Epigenetic oscillations. . . ......... . ... . .. . ............. .. . ............ 248
Endogenous oscillations. . .. . ........ ... . .... ... . .... .. ..... .. ......... 249
�Muscular contraction.. ............... ................................ 249
Periodic membrane transport. . . . .............. ...... ......... ...... . .. 249
MODELS AND THEORIES. .. . ... .. .. .. . . ... . ... .. .... . .. ...
. . . . . . . . . . • • • . 250
Chemical models. .... ..... ..... .. .. ...... .. .........................
. 250
Specific mathematical models. . . ........ ..... .. .. .. . ..... .. .. .......... 251
General mathematical models. .. . ... .. . ...... ... ... . .......... .... ...... 251
CONCLUSIONS AND OUTLOOK. . .. . . ... . .... ...... .... .......... .. ..... . .. 252
DEFINITIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . .. 254

INTRODUCTION

Scientific interest in rhythmic phenomena in nature can be traced back

to a report written by Z inn, "Von dem Schlafe der Pflanzen," published in
1759 (1). Early scattered interest together with later systematic studies by
biologists led to the development of a vast field, concerned principally with
the description and interpretation of longer periodic rhythms of complex
physiological functions in numerous systems of plant, animal, or human ori­
gin. These studies gave rise to various new de finition s of periodic phenom­
ena, such as diurnal rhythms and circadian clocks. Furthermore they led to
the realization that longer periodic rhythms play a significant role in the
regulation of the activities of nearly all biological systems (for review see
2-6). While this type of periodicity is out of the scope of this review, bear
1 This review covers the present-day knowledge and our major viewpoints of the

field, and does not represent all the opinions of other investigators. The area of
electrogenic and neural oscillations is not discussed. The survey of the literature
for this review was concluded in October 1970.
237
 238 HESS & BOITEUX

in mind that oscillatory phenomena in biochemistry may differ from biologi­

cal rhythms only in their degree of complexity and their frequency, and that
mechanistic relationships between both organizational levels will certainly
be elucidated in the future. This review is restricted to oscillations in sys­
tems of homogeneous cell and subcellular systems which can be defined in
biochemical terms and traced to a molecular level. Oscillations of compo­
nents in neural systems will not be covered ( for review see 7-9). Defini­
tions that may be useful to the reader are given at the end of the review.
Historically, it is of interest to trace attempts to reduce biological peri­
odicities to simple chemical systems. Early in 1910 Lotka, in a pioneering
study based on a law of mass action, analyzed the kinetic conditions in ho­
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mogeneous and heterogeneous systems leading to oscillations (10, 11). In

1921 Bray (12) discovered a periodic chemical reaction involving a cata­
lytic decomposition of hydrogen peroxide by iodine in a homogeneous solu­
tion. In 1943, analyzing the physicochemical basis of electrogenic rhythmici­
ties with nerve models, Bonhoeffer (13, 14) drew attention to the general
conditions necessary to generate periodicities in complex homogeneous and
heterogeneous systems in a series of papers, HUber periodische chemische
Reaktionen." He stressed the concept of two independent variables in terms
of negative and positive cross-coupling of two reaction pathways and the
autocatalytic mode of their kinetic characteristics, and formalized their limit­
cycle behavior. Later, in 1954, Teorell pioneered the field of membrane-bound
oscillations with model experiments on the rhythmic properties of the e1ec­
troosmotic flow passing through coarse membranes. In this study he ob­
served oscillations of the potential and volume of flow at constant current
(15, 16).
The experimental field of biochemical oscillations was opened by Wilson
& Calvin in 1955 (17) who demonstrated oscillatory transients of the pho­
tosynthetic cycle. A detailed kinetic theory of this phenomenon was ad­
vanced in 1958 by Chernavskaya & Chernavskii (18) on the basis of a hy­
pothesis of the self-oscillating mode of the dark reactions of photosynthesis.
The authors presented a system of chemical kinetic equations describing the
Calvin cycle and analyzed its general properties in terms of the "character­
istic plane" and "field of isoclines" as well as its limit-cycle behavior. They
suggested that "most of the biological processes either occur in a self-oscil­
lating state, or are near to it."
Whereas earlier observations in various laboratories indicated instabili­
ties in glycolysis, clear oscillations of NADH were reported by Duysens &
Amesz in 1959 (19) and later in yeast cells by Ghosh in the laboratory of
Chance (20). This observation was followed by extraction and isolation of
the oscillating glycolytic system from yeast cells ( 21, 2 2), demonstration of
continuous glycolytic oscillations in a cell-free system, and elucidation of
the underlying mechanism (23, 24).
This development coincided with the observation of oscillatory ion
movements in mitochondria (25, 26), where different metabolic functions as
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 239
well as components of the respiratory chain displayed sustained periodic be­
havior (27, 28). Here further studies of the complex phenomenon led to the
disclosure of distinct control properties of the inner mitochondrial mem­
brane (29). These two examples demonstrate that the oscillations in com­
plex biochemical systems can be reduced to thc functional properties of en­
zymes or membranes and can finally be recovered in structural changes at
the molecular level. Indeed, oscillations in experimental systems reflect the
complexity of the system in structure and time. Thus, the detection of oscil­
lations provides an excellent opportunity to study the dynamic behavior of
cellular and subcellular systems.
There is no review article on oscillatory phenomena in biochemistry at
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present. The subject has been discussed in detail in a symposium held at

Puschino na Oke in 1966, published in (30). The proceedings of two inter­
national symposia on biochemical oscillations and membrane instabilities,
held at Prague in 1968 and at Hango in 1969, will soon be published in book
form which summarizes the papers presented by various authors (see 9,
31).

SOLUBLE OSCILLATORS
Enzymes.-The first clear oscillatory reaction in a soluble enzyme sys­
tem was observed in an open system of horseradish peroxidase and lactoper­
oxidase in the presence of a reductant and oxygen. The horseradish peroxi­
dase system oxidizing both pyridine nucleotides (32-34) and indoleacetate
or dihydroxyfumarate (35,36) oscillated with a rather limited stable range
between ferriperoxidase and compound III in phase with the oxygen con­
sumption. Oscillations were favored by an acid pH (33). Chemilumines­
cence oscillations were also observed (35). With la ct operoxidase at neutral
pH in presence of NADPH and oxygen,stable oscillations between the fer­
ric state and compound III over more than 50 cycles were observed, with
frequencies of approximately 0.2 min-1 and amplitudes of approximately 0.5
extinction units at 418 nm (see 31 ) . The system was fortified with glucose
6-phosphate dehydrogenase and glucose-6-P to foster NADPH regenera­
tion, 2,4-dichlorophenol to stimulate the pyridine nucleotide oxidation rate,
and methylene blue to accelerate the decomposition rate of compound III.
The system involved at least 10 reaction steps, including the peroxidase cy­
cle and a chain reaction coupling the oxygen uptake to the formation of
compound III (33). On the basis of the differential response of compound
III to the addition of various donors such as NADH, indoleacetate, and
dihydroxyfumarate, a direct function of compound III as a regulatory com­
ponent in the oscillation circuit has been questioned (35, 37). The system
has a clear feedback structure and displays oscillation and bistability (36)
if a steady state is induced by generating a steady donor and oxygen supply.
On the basis of an autocatalytic mechanism, the experimental observations
can be fitted to the Lotka model (37).

A report has been published on oscillations of the activity of purified

 240 HESS & BOITEUX
creatine kinase in buffer solutions depending on the enzyme concentration
and the state of its thiol groups (38). The nature of these oscillations has
not yet been elucidated.

Glycolysis in yeast eztracts.-Oscillations of the NADH level, first re­

ported in intact yeast cells (see Celullar Systems), have also been demon­
strated in cell-free extracts of yeast (21, 22). This achievement has greatly
facilitated investigations on the mechanism of the oscillations. Oscillating
glycolysis is the best-understood oscillating system today.
Single additions of maltose or trehalose readily induce NADH oscilla­
tions in the extract (23, 24, 39), while additions of saccharose, glucose,
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or fructose do not. However, continuous oscillations over many hours can

be obtained with these substrates and also with hexose 6-phosphates by us­
ing a substrate injection technique (40-43). Oscillations are also induced by
phosphate if the system is initially saturated with substrate but phosphate­
limited (42). The requirement for the generation of oscillation is a constant
and relatively low input rate of glycolytic substrates, maintained in the case
of maltose and trehalose additions by a low activity of the respective disac­
charases in the extract (44).
The amplitUde of the NADH oscillations is in the range of 10-4 M(20).
Spectroscopic studies indicate that the prominent component recorded by
fluorimetry or spectroscopy is a protein-bound NADH with maximum ab­
sorbency at 355 nm (4S). Amplitude, waveform, and frequency can be var­
ied by changing the injection rate of the substrate (41, 46). Continuous and
modulated sinusoids, single and double pulse s, strong asymmetric cycles,
square wave approximations, and spikes have been obtained with frequen­
cies between 0.05 and 0.5 min-1 and well over 160 continuous cycles (23, 24,
41, 57). The temperature-dependent frequency indicates an activation en­
ergy of 19 kcal X mole-1 from 6-33°C (29).
Studies on the metabolic balance during glycolyti c oscillations demon­
strate a complete recovery (96%) of the amonnt of substrate turned over
into glycolytic intermediates and the endproducts (43). Equally well bal­
anced are energy production and consumption during one cycle, due to AT­
pase activity and the accumulation of fructose- l,6-diP (43). Detailed inves­
tigations of the kinetics of glycolytic intermediates show oscillations of the
levels of all metabolites with concentrations ranging between 10-5_10-3 M
(42). In addition an oscillation of pH and a pulsed production of carbon
dioxide is recorded (40, 41).
A series of studies were carried out to locate the control points along the
glycolytic sequence which are responsible for the generation of oscillations
(42, 43, 47, 48). Analysis of the kinetic behavior of the glycolytic compo­
nents relative to each other led to a pattern as illustrated in Figure 1, where
the normalized concentration changes of the glycolytic intermediates per
unit time are summarized (42, 43). Concentrations of metabolites change
during the oscillatory state with equal frequency, but with differing phase
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 241

angles re l ati ve to each other. According to their time-dependent change of

concentration, the glycolytic metabolites fall in two groups. In each group
maxima and minima of the concentration changes coincide in time. The two
groups differ by the phase angle �a. It is important to note that,Aa can be
varied, depending on the experimental conditions (42, 43).
The location of the control points of the oscillation can be demonstrated
with the help of the crossover diagram plotted in Figure 2. Here the phase
angle between adjacent glycolytic intermediates is plotted as a function of

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FIGURE 1. P hase shift of osciI1ating glycolytic intermediates. The amplitude of
the concentrations is normalized (31, 42).

their respecti ve location in the glycolytic sequence. A pha se shift by 1800 is

found between the metabolites fructose-6-P and fructose-l,6-diP as well as
between phosphoenolpyruvate and pyruvate indicating the enzymes phos­
phofructokinase and pyruvate kinase as control points. Furthermore, the re­
action step between glyceraldehyde-3-P and 3-phosphoglycerate catalyzed
by the enzymes glyceraldehyde-3-P dehydrogenase and phosphoglycerate ki­
nase corresponds to the variable phase angleAa. Since all control points
are coupled to the adenosine phosphate system taking part either in ATP
synthesis or in ATP breakdown , the ATP system obviously is responsible
for the propagation of the oscillation along the chain. The extent of the
phase shift demonstrates that phosphofructokinase and pyruvate kinase are
the strongest control points of the system while the smaller phase shift be­
tween glyceraldehyde-3-P and 3-phosphoglycerate indicates a comparably
 242 HESS & BOITEUX

weaker control of the enzymes glyceraldehyde-3-P dehydrogenase and phos­

phoglycerate kinase (42, 43).
Evidence for this mechanism is based on additional experimental data.
The controlling function of phosphofructokinase can be directly demon­
strated by testing t he ability of various glycolytic substrat es to initiate gly­
colysis and oscillation.
Fructose-6-P is the last metabolite within the glycolytic reaction se­
quence that will induce oscillations, while fructose-l,6-diP, the next inter­
mediate in the sequence, will produce only carbon dioxide but no oscillations
of any metabo li te (41, 43). Further evidence is given by the dynamic re­
sponse of the oscillating yeast extract to additions of glycolytic enzymes,
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intermediates, and different ions (42, 47-51). All substances interfering

·
90 GAPDH/PGK

OO���-H��____+-__�
CAP
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GAP
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-450

FIGURE 2. Phase-angle shift of glycolytic intermediate concentrations during

oscillations in a crossover plot (31, 42).

with the balance of the adenosine phosphate system cause variations of am­
plitude, frequency, or waveform of the oscillations. In particular the adeno­
sine nucleotides exert strong direct control upon the phase position of the
components of the oscillating system (43, 47, 48). In addition, the action of
glycolytic inhibitors such as iodoacetate, fluoride, or substrate analogs like
deoxyglucose support this mechanism (21, 52).
The basic feature of this mechanism is the assignment to specific en­
zymes of a definite control function responsible for the oscillatory dynamic
of the whole process. Detailed investigation of the activities and dynamic
properties of the glycolytic enzymes under conditions of oscil1ation demon­
strates a mean activity in the range of 2-30% of their respec tive maximal
en zyme activity (42). In particular, phosphofructokinase changes its activ­
ity at least by a factor of 80-90 during one oscillatory period (42). Fur­
thermore the enzymes, which exhibit dynamic control, also show allosteric
properties: phosphofructokinase (42, 43, 53), glyceraldehyde-3-P dehydrog­
enase (54), pyruvate kinase (55), and pyruvate decarboxylase (56). In­
deed, these enzymes provide a structure with inherent dynamic kinetics
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 243

which can readily produce instabilities or, coupled within the feedback
structure of the glycolytic system, oscillations of the glycolytic flux.
Additional evidence for the validity of the mechanism of glycolytic oscil­
lations is presented by reconstruction experiments which demonstrated that
glycolytic oscillations are due only to the essential components of the glyco­
lytic system (40, 41). In these experiments, oscillations of square wave
character as well as a spikelike waveform with a rise time of less than 1.5
sec and approximately 1 min intervals have been recorded (41).
In summary, glycolytic oscillations are generated by feedback activation
and inhibition of the allosteric oscillophor phosphofructokinase coupled by
the adenosine phosphates to the other kinases and the ATP-consuming sys­
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tems. The periodic changes of the flux of glycolytic intermediates produced

by this system are coupled stoichiometrically to the NADH/NAD system by
the enzymes glyceraldehyde-3-P dehydrogenase and alcohol dehydrogenase.
This structure is a double-crossed network with feedback loops in both the
adenine and pyridine nucleotide circuits.
A comparison between glycolytic oscillations in yeast extracts and in
suspensions of intact cells (see Cellular Systems) indicates no difference in
the mechanistic principle of either system. The higher frequency of the lat­
ter system may well be caused by transport-restricted substrate supply to­
gether with the high rate of ATP-consuming reactions in the cell. This is h�
agreement with the observation of the Harden-Young effect in oscillating
extracts, leading to a gradual accumulation of fructose-l, 6-diP due to the
imbalance of the rates of ATP synthesis and breakdown.
The physiological significance of the glycolytic oscillations cannot be
evaluated at present. In principle, their property is inherent in feedback
structure of the glycolytic pathway and its control mechanism. Since spon­
taneous NADH oscillations, induced by glycolytic substrates, are observed
in yeast-cell suspensions (39) and even in a single yeast cell (57), the gly­
colytic oscillations in yeast are clearly a property of the physiological state
of the cell.

Oscillating glycolysis in other systems.-Glycolytic oscillations have also

been observed in aerobic suspensions of Ehrlich ascites tumor cells (58)
and in beef heart extracts (59). Whereas the generator of oscillations has
not been clearly located in the first case, the importance of phosphofructoki­
nase as the oscillophor of glycolysis in beef heart extracts has been stressed
on the basis of detailed metabolite assays, and by modifier studies. The os­
cillations are restricted to a rather small pH range ( 60-62).

MITOCHONDRIAL SYSTEMS
At the 1965 FASEB meetings two reports (25, 26) described a new phe­
nomenon: oscillations of light scattering and ion .fluxes in suspensions of
mitochondria. The oscillations were induced in both cases by adding iono­
phoretic antibiotics. Valinomycin ( 63-65) was used to activate the potas-
 244 HESS & BOITEUX

sium transport in rat liver mitochondria, and the transport of monovalent

cations was stimulated by actin homologs (66-68). Subsequently oscillatory
phenomena in mitochondria have also been found without use of antibiotics.
The following oscillations have been described: gramicidin-induced swell­
ing-contraction cycles (69), respiration-linked rebounds, oscillatory move­
ments of Ca++ and H+ between mitochondria and medium (70, 71), and
cyclic volume changes under conditions of energized ion transport by
EDTA-t reated mitochondria (27, 72). Mg++ in concentrations greater than
EDTA prevented the volume oscillations. Mg++ decreases the permeability
of the mitochondrial membrane to monovalent cations while chelating
,

agents such as EDTA or citrate act oppositely (73, 74). Thus, enhanced
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transport activity of the mitochondrial membrane is common to all oscillat­

ing mitochondrial systems reported so far. The dominating role of mem­
brane permeabilIty is stressed by the fact that frequency, amplitude, and
damping of valinomycin-induced oscillations respond to variation of the ca­
tion transport rate, irrespective of the mode of variation (28).
Both types of mitochondrial oscillations, the antibiotic-induced and the
nonionophor-supported, have some basic conditions for their generation in
common: the presence of a monovalent cation, a permeant anion, preferably
phosphate, and substrate or ATP. In both, swelling of mitochondria depends
on an energy source, and recontraction occurs when the energy source is
blocked (67,72).
In the early reports of mitochondrial oscillations only two or three cy­
cles with rapidly decreasing amplitudes were recorded. Subsequently, a long
train of virtually undamped oscillations of H+ and K+ concentrations in a
suspension of pigeon heart mitochondria has been demonstrated (75). The
phenomena were quantitatively analyzed in terms of amplitude, frequency,
damping factor, and the K+jH+ ratio. The frequency of concentration
changes in the two ions is the same and - 1 min 1 ; also their phase relation
-

is constant at 1800• When potassium ions enter the mitochondria, protons

are extruded, and vice versa. The ratio of K+ to H+ movement is about 4 or
even higher, depending on the concentration of the permeant ions and pH
( 73,75,76).
From the unequal cation exchange one can expect anions and water to
exchange across the membranes as well, thus balancing the electroneutrality
and osmolarity of the system, as monitored by light scattering (77). Mito­
chondria can be trapped in different oscillatory states by a density gradient
-

technique for rapid fixation of mitochondria by glutaraldehyde (78). Elec­

tronmicrographs of such preparations show morphological changes in the
mitochondria (79), whose swollen and contracted states resemble the ortho­
dox and condensed forms, previously described for mitochondria of differ­
ent metabolic states (80, 81). By correlating packed volume measurements
with the e1ectronmicrographs it has been shown that the mitochondrial vol­
ume changes reflect changes of the sucrose-impermeable space of the mito­
chondrion (82). This finding has been confirmed by the isotope dual label
-
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 245

technique (83, 84). The mitochondrial matrix space can increase during os­
cillations by some 40% due to ion translocation and osmosis (82).
Concomitant with the oscillatory ion movements and volume changes of
mitochondria, periodic variations of the redox state of respiratory carriers
can be recorded. Oxidation-reduction cycles of pyridine nucleotides (28, 72,
77), flavoproteins (28, 85), and cytochromes (28, 85, 86) have been rc­
ported as well as changes in the rate of oxygen uptake (25, 72, 77). Under
reduced oxygen tension an oscillating respiration rate has been demon­
strated directly (87).
The multiplicity of oscillating parameters complicated an investigation
of the basic mechanism of mitochondrial oscillations. All parameters ana­
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lyzed so far oscillate with the same frequency, but with distinct phase shifts
relative to each other (d Figure 3). Additions of ADP and ATP to sub­
strate-fed oscillating mitochondria shift the whole set of parameters to the
positions indicated, clearly demonstrating control functions of the energy­
coupling mechanism. A crossover point between flavoproteins and cyto­
chrome b reveals a cyclic change between energy and substrate control dur­
ing oscillations (28, 85). Indeed, substrate control by competitive anion
transport, together with energy-dependent transport of the respective ca­
tioH, is in accord with the requirements for the generation of oscillations as
outlined above. Interesting speculations have been presented on the general
aspect of metabolic control by energy-dependent cation transport (88).
Further information on the mechanism of oscillation is given by titration
with ions. These experiments reveal a strong positive feedback control of
hydrogen and potassium ions on the oscillating system (cf Figure 3). Addi­
tion of either of these ions to the suspension medium results in a phase shift
of all oscillating parameters to the indicated positions and additional ejection
of the titrated ion from the mitochondrial matrix space. The lowest effective
concentration of either ion is in the range of its oscillatory amplitude. Thus,
mitochondrial oscillations are controlled by positive feedback coupling of
ion fluxes to the respiratory system, and pH and pK differentials are identi­
fied as synchronizers for the synchronous oscillations of the mitochondrial
population (28,89).
The dependency of the membrane function upon ion differentials
strongly suggests a charge-coupled structural change of the mitochondrial
inner membrane. This idea is supported by optical rotatory dispersion and
circular dichroism studies on mitochondria, indicating a correlation of vol­
ume changes with changes in secondary structure of inner membrane pro­
teins during oscillations (29). Furthermore the high activation energy of
19-26 kcal for a change of frequency (75,77) excludes a simple chemical re­
action sequence. The dominating role of membrane properties for the mito­
chondrial oscillations is stressed by the dependency of the oscillation fre­
quency on the transport rate of potassium ions (28,89) and by the damping
effect of local anesthetics (90).
There has been some speculation with respect to the natme of energy
 Respiration Rate

*
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::r::
t:t:1
(f]
(f]
f(;O
td
o
,....,
0--3
�
�

Redox State of Cytochrome b

len Transport

Phase Angles obtained by Titration

FIGURE 3. Phase relations in mitochondrial oscillations and shift of phase angle by titrations. Outer shells: Phase
relations of volume change, ion transport, respiration rate, and redox state of cytochrome b. Central part: Phase an­
gles obtained by additions of H+, OH-, K+, ADP, and ATP to oscillating mitochondria.
Following the ai)dition of the respective compound, the phase angle at the time of addition is rapidly shifted to
the indicated position, while the oscillation continues with unchanged frequency (with minor modifications from 28,
89).
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 247
coupling (91) or the mode of action of uncouplers (92) on the basis of
mitochondrial oscillations. In addition to the short-period oscillations de­
scribed so far, cyclic changes of mitochondrial respiration with a period of
about 30 min have been reported (93).

CELLULAR SYSTEMS
Metabolic osc illat ions. T here are a few reports on oscillating parame­
-

ters in single cells that can be traced to their metabolic origin. Pyridine
nucleotide oxidation-reduction cycles with periods of - 40 sec have been
observed microspectrophotometrically in individual yeast cells (57). The
membrane potential of single muscle fibers of frog skeletal muscle oscillates
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with periods in the minute range as revealed by autocorrelation analysis

(94). The former cycles are clearly caused by oscillating glycolysis (cf Sol­
uble Systems), while for the latter the same origin is probable but not yet
established.
Oscillations of the transmembrane potential difference are induced in the
alga Hydrodictyon reticulaturn by switching from light to dark or vice
versa (95). The measured potential changes were - 40-50 mV with a pe­
riod in the minute range. Addition of valinomycin to the medium resulted in
an increase in the number of recorded cycles from 4 to 14. The close rela­
tion of chloroplasts and mitochondria with respect to ion and energy trans­
port st rongly suggests that the recorded oscillations of the membrane poten­
tial are caused by periodic ion movements across the chloroplast membrane
in analogy to the oscillating mitochondrial system (see above).
Indeed, the photosynthetic apparatus is susceptible to oscillations. In ad­
dition to the observations related above, oscillations in oxygen evolu­
tion are induced in Chlorella pyrenoidosa by changes in white illumination,
when a low CO2 concentration is maintained or in the presence of poisons
known to inhibit the photosynthetic carbon cycle. The period of the oscilla­
tions varies between 4 and 60 sec with amplitu des of less than 1 p.1 02/ml
cells X hr, about 1 % of the constant steady-state range. The mechanism of
this oscillation is still obscure (96).
The majority of oscillatory phenomena is observed in cell suspensions
and cultures. This fact implies the existence of a synchronization effect be­
tween the individual cells of the population. A synchronous metabolic rate
of a given population is often initiated by sudden metabolic pertu rbation ,

such as aerobic-anaerobic transition (20,97-99) or addition o f oxygen (72,

87) or substrate (39,72,97-99),forcing the individual metabolic systems of
the population to identical states. Examples of this type of triggered oscilla­
tions in cell suspensions are the glycolytic oscillations of yeast (39) and the
oxidation-reduction cycles in NADH of Klebsiella aerogenes (107). Both
systems are capable also of spontaneous oscillations. Synchrony of metabo­
lism often can be maintained over long periods; in the case of yeast cells,
for many hours (39, SO, 108). This raises the question of a synchronizing
metabolite (cf Mitochondrial Oscillations). In favor of such a compound
 248 HESS & BOITEUX

are the results of experiments where two populations of yeast cells, oscillat­
ing with different phase angles, are mixed and the resulting phase shift of
the oscillating mixture is recorded. The synchronizer, however, has not yet
been identified (50).
Triggered and spontaneous oscillations can be found in yeast cells grown
aerobically or anaerobically on different substrates and harvested in their
logarithmic or stationary growth phases (39). The oscillatory response has
been observed following continuous and discontinuous additions of mono­
and polysaccharides such as glucose, fructose, sucrose, maltose, and treha­
(39).
lose to the cell suspension
The amplitude of NADH oscillation accounts for a change in its cyto­
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solic concentration of about 25 p.M (20). In contrast to oscillations in cell ­

free extracts, the waveform has a clear sinusoidal character in intact cells
(20 , 39), and the frequency is somewhat higher, ranging between 1-3 min-l.
The frequency variation of the oscillation during a glucose injection ex­
periment was 3 .5% over a 50 cycle range (39). The mean frequency and
damping f actor depend on t he nature and inject io n rate of the substrate.
The temperature-dependent increase of frequency between 19.50 and 400 var­
ies by a factor of 4-2.2 for a 100 interval (20 , 100). There are no major
differences in the mechanism of the glycolytic oscillations in yeast cells and
yeast extracts (97-106, see also above).

Epigenetic oscillations Osci llations of biosynthetic systems result from

.-

interference with the control mechanism of protein synthesis. The oscilla­

tion of the rate of synthesis of NAD-dependent glutamate dehydrogenase in
Saccharomyces cerevisiae R59 was rep orted ( l09). The oscillations with a
f requency of approximately 0 .15 hr-1 are highly damped and triggered after
derepression of exponentially growing cells with glutamate as single source
of nitrogen. This involves DNA and messenger RNA as essential parts of the
oscillating circuit. Furthermore, oscillations of ornithine transcarbamylase
in Escherichia coli after derepression by removal of arginine have been de­
scribed (110). The rate of ,B-galactosidase synthesis was f ound to oscillate
after initiation by a transition from glucose to lactose as the carbon source,
with a period of about 50 min in various strains of E. coli under conditions
of asynchronous growth, indicati ng a partially autonomous mechanism in­
volving the complete circuit of the system of protein synthesis (111). Also,
the specific activity of carnitine dehydrogenase of Pseudomonas aeroginosa
shows damped oscillations induced by L-carniti ne (112).
Periodic changes of metabolic functions are found also under conditions
of continuous cell culture. A number of parameters such as pH, redox state
of respiratory carriers, and respiration rate are reported to oscillate in K.
aerogenes cultures (113). Periodically changing metabolic rates are coupled
to the cycle of cell division in yeast (114), and the same has been reported
for pyruvate formation and ,B-galactosidase activity in synchronously grow­
ing continuous cultures of E. coli (111, 115). The periodi c activity changes
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 249

of thymidine kinase in Physarum polycephalum are also clearly synchronous

to the mitotic cycle of this fungus (116).

Endogenous oscillations.-In contrast to these examples an endogenous

circadian rhythm, independent of the nuclear function, has been observed in
the oxygen balance of the unicellular green alga Acetabularia mediterranea
(117). The now classical studies on Gonyaulax oscillations reveal different
systems which follow a diurnal period: a luminescence flash oscillation, a
luminescence glow oscillation, a cell division oscillation, and an osciIlation
of the photosynthetic capacity (118-121).
Current literature includes an increasing number of reports on short-term
Annu. Rev. Biochem. 1971.40:237-258. Downloaded from www.annualreviews.org

or circadian rhythms in cellular systems, which are obviously of biochemical

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nature, but at the time still poorly understood in their mechanisms. Most of
them may be coupled to a differentiation process or the life cycle of the
species.

Muscular contraction.-Slow fluctuation of the spontaneous activity of

tonicity of isolated Taenia coli of guinea pigs with periods in t he range of
minutes is reported to depend on metabolism, but is not understood biochem­
ically (122, 123). A detailed analysis of mechanically induced oscillation of
glycerol-extracted ATP-supported insect fibrillar muscle revealed a mecha­
nochemical coupling mechanism as the basis of the muscle contraction cycle
of living insects (124, 125). A forced change in fiber length is followed by
increased fiber tension with a frequency-dependent delay. It has been found
that stretching-release cycles of the isolated fiber above a threshold fre­
quency lead to an increasing actin-myosin interaction rate associated with
an increasing rate of oscillatory power output and ATP splitting by the ac­
tin-activated ATPase. The rate of energy converted into mechanical works
is optimal under physiological conditions at a frequency of 20 Hz and an
amplitude of 2% fiber length. The experimental value of 105 ergs/sec per
g muscle compares very well with 3 X 105 ergs/sec per g in living beetle
muscle. Under experimental conditions about 50% of the free energy of
ATP-splitting is converted into mechanical work (126). The same type of
mechanical activation of the contractile system has been found in extracted
fiber of skeletal muscle which oscillates during sinusoidal length changes of
0.5% at 5-10 Hz (127). These observations are currently related to the
molecular properties of the contractile system. Snol et al (see 30) observed
synchronous oscillations of ATPase activity, the amount of titrable SH
groups, and the absorption properties of solutions of actin, myosin, and
actomyosin, which are interpreted as results of conformational oscillations.

Periodic membrane transport.-A discussion of the large field of peri­

odic transport processes across membranes without accompanying chemical
reactions is out of the scope of this review. The reader, however, should be
aware of the formal and original relations between the electrogenic and ion-
 250 HESS & BOITEUX

ogenic oscillations in various systems such as conducting nerve fibers or

acting musele cells.

MODELS AND THEORIES

Chemical models.-Examples of periodic chemical reactions in homoge­

nous solution may be considered as models for oscillations in biochemical
systems. The reaction between hydrogen peroxide and iodate in dilute sul­
furic acid is autocatalytic and delivers oxygen with a rate p eriodi c with
time (12). Studies based on the rate of oxygen evolution suggest a control
of catalytic intermediates in a feedback loop by the endproduct oxygen
Annu. Rev. Biochem. 1971.40:237-258. Downloaded from www.annualreviews.org

(128). Different results, however, were obtained in a detailed study on the

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kinetics of iodine formed as an intermediate in the complex reaction se­

quence. Oxidation of iodine by hydrogen peroxide is assumed to proceed by
a branched free radical chain resulting in oscillation (129). This interpreta­

tion is in agreement with nonsinusoidal oscillation, which appears to be of

the relaxation type. Furthermore, di g ital computer calculations based on the
branched-chain mechanism produced concentration-time curves very similar
to the experimental ones (130).
Another example is the oxidative decarboxylation of citric or malonic
acid by bromate as catalyzed by cerium ions ( 131, 132). In a quasi-open sys­
tem some hundred oxidation/reduction cycles of cerium ions can be recorded
with a frequency of (H).6 min-1, depending on the experimental conditions.
The effect of bromine derivatives of malonic and acetic acids on the initial
nonoscillatory phase of the reaction led to the assumption of a reaction se­
quence involving feedback inhibition (133). The rate of autocatalytic oxida­
tion of cerous ions is controlled by the strong inhibitory action of dibromo­
malonic acid and the release of inhibition due to decarboxylation.
The malonic acid-bromate system catalyzed by cerium ions has been re­
ported to display under appropriate conditions "steady spatial waves" (134,
135), a phenomenon predicted by the theory of the thermodynamics of irre­
versible processes (136), but not yet observed in biochemical systems.
Heterogeneous chemical and electrochemical reactions such as the passi­
vation-activation behavior of metallic electrodes in acids have frequently
been used as models for biological excitation processes, and especially nerve
models have been developed on this basis ( for review see 137). Whereas
these systems cannot be discussed here, we would like to stress the proper­
ties of artificial membranes. Experimental bimolecular lipid membranes
have been reported to develop electrokinetic phenomena undistinguishable
from cellular action potentials and include thresholds, bistable flip-flop ki­
netics, and spontaneous rhythmic firing (138-141). The permeability of the
membranes for ions can be modified by local anesthetics or ionophoretic an­
tibiotics such as valinomycin or l1onactin. These properties of artificial
membranes show striking similarity to natural membranes of the nerve
axon or the mitochondrion that make them excellent experimental models to
study the complex function of biological m embranes .
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 251
SPecific mathematical models.- Mode1s of electrochemical and pure
chemical systems have been analyzed by a number of authors (132-134,
142), each of them deriving conclusions about the kinetic structure of oscil­
lating systems in general. The single enzyme system, horseradish peroxidase
catalyzing an oxidation reaction in an open system (see above), was inves­
tigated in detail with a mathematical model based on Lotka's original auto­
catalytic structure (129).
Specific models of glycolytic oscillations have been developed and ana­
lyzed by analog and digital computer studies. Several models are based on
the complex kinetic properties of phosphofructokinase, which plays an es­
sential role in the generation of glycolytic oscillations as reviewed above. A
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simplified model (143) representing a minimum structure of glycolysis sim­

ulates oscillations with a first-order feed-in step, an enzyme step activated
by its product, and a second enzyme step removing the activating compound.
Another simple model (144), based on an enzymic reaction involving sub­
strate inhibition and product activation in an open system, explains qualita­
tively many data on the single frequency oscillation in glycolysis. Both mod­
els have been varied and extended in order to meet the gap between calcu­
lated properties and experimental data (145, 146). A detailed model of gly­
57 si­
colytic oscillations in the heart supernatant preparation, consisting of
multaneous differential equations representing 101 chemical equations, was
developed and analyzed by the digital computer technique (147, 148).
Models of more complex oscillating systems have also been presented:
an equation system describing the reactions of photosynthesis (18), a sys­
tem simulating the regulation of synthesis of f1-galactosidase (31, 149, 150),
a mechanism leading to the oscillation of respiratory rate in K. aerogenes
( 151), and general systems describing enzyme synthesis (152, 153). A sum­
mary of models dealing with the dynamics of membrane processes is given
elsewhere (154, 155; see also 31).

General mathematical models.-The increasing number of experimental

data on oscillating biochemical reactions led to attempts to develo p g ener al
models of dynamic systems capable of oscillations. The simplest approaches
tried to vary the autocatalytic reaction mechanism and the mechanisms of
self- and cross-coupling interactions of a multicomponent pathway. Espe­
cial ly the latter case was studied with respect to kinetic str uctures yiel ding
sustained and transient oscillations (145, 156). The type of feedback loop
and the number of individual enzymatic steps, as well as their reaction or­
der, significantly influence the type of oscillation and its limit-cycle behavior
(145, 157, 158). In addition, the frequency dependencies (145, 157, 159),
(160-162), and the projection of oscillation from a pri­
stability properties
mary oscillation source along a chemical pathway have been analyzed
(145). Fur thermor e, the behavior of an assembly of weak individual oscilla­
tors has been studied with respect to coupling and synchronization (163).
Latest models of oscillating systems deal with spatial oscillations of chemi­
cal reactions as well as with th e time-space problem (135, 136).
 HESS & BOITEUX

Special attention has been paid to the thermodynamic structure of the

system and its capacity to display oscillations. Closed systems, being de­
scribed by linear or nonlinear differential equations, cannot oscillate when­
ever they approach the equilibrium state ( 164, 1 65 ) . In contrast, compo­
nents of an open chemical system can oscillate continuously around stable or
unstable steady states if the system is sufficiently far from equilibrium, es­
sentially irreversible, and outside the range of linear thermodynamics of
irreversible processes. A cross-coupled structure of the components is also
required ( 136, 166, 167) . Indeed, a time organization as displayed by o�cil1a­
tions is a response to "far from equilibrium conditions" in the same way as
dissipative structures are ( 1 67) . Systems of this type can generate "sym­
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metry-breaking chemical instabilities," which may be important in biological

systems es� ecially since such instabilities might lead to "symmetry breaking"
with respect to diffusion ( 166 ) .

CONCLUSIONS AND OUTLOOK

On the basis of the results of experimental and theoretical studies, a

number of kinetic and thermodynamic criteria for the occurrence of oscilla­
tions can be summarized :
1. The kinetic strttctttre of the simplest system capable of oscillatory mo­
tion is based on autocatalysis. More complex systems show simple and mul­
tiple types of feedback structures. The kinetic characteristics of individual
reaction steps of such a system can vary. However, their combinations must
generate an ordering of power, which ensures amplification as well as inhi­
bition to allow the system to settle in an oscillatory state. These systems can
be described by a set of nonlinear differential equations and display limit­
cycle behavior.
2. The thermodynamic requirements for a generation of oscillatory
states are based on the conditions that the classical Onsager relationships do
not hold and the system is open. Thus, the steady state must be maintained
sufficiently far from thermodynamic equilibrium, providing a minimum rate
of energy dissipation.
3. The interacting molecular structures in oscillating systems produce a
kinetic behavior of higher order, by either stoichiometric or catalytic inter­
action. In both cases the property of cooperativity is involved which yields a
graded response and "all or none" action. Examples of cooperativity are the
interactions between nucleic acids, the cooperativity of membrane compo­
nents ( 1 69 ) , and finally the allosteric transitions of enzymes. It is interest­
ing that glycolytic oscillations and mitochondrial oscillations are bound to
the function of cooperative structures. It might be suggested that oscillatory
phenomena in biology will finall y be based on this very property of coopera­
tivity of molecular structures.
4. The time scale reported in oscillating systems covers the whole time
range relevant to living systems. The frequencies stretch almost continu­
ously over ten orders of magnitude, reflecting the temporal organization of
 OSCILLATORY PHENOMENA IN BIOCHEMISTRY 253
the living world. It is interesting to see that the biochemical oscillations sub­
j ect to this review fill the gap between the circadian oscillation and the
neural frequencies observed so far in biological systems.
5. The Physiological significance of oscillations is frequently questioned.
The answer must be that oscillations are a property of complex dynamic sys­
tems, as predicted by the theorem of Rubin & Sitgreaves ( 168 ) . Indeed, on
every level of its organization biological systems are highly complex and
dynamic structures, tightly controlled by multiple feedback interactions.
Therefore, one has to expect oscillations to occur in all biological systems as
is demonstrated by the examples outlined on cytoplasmic, mitochondrial, ep­
igenetic, cellular, and population levels. In addition, the following aspects
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must be considered.
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6. Oscillations around a steady state might add an increasing stability to

a process and also function as a synchronizing clock ( 170-172 ) , which is

important e.g. in the timing of large systems such as the mass of the heart
muscle. Recently it has been suggested that the synchronizing function of
oscillations might well operate as a mode which adds positional information
to a cell during development and regeneration. The structure of differenti­
ated tissue is the result of distinct spatial and temporal organization of the
deVeloping processes, and questions arise as to how the time is read and
how each locus within the cell mass receives information on its position.
The model suggests that the positional map is given by oscillatory propaga­
tion of activity and concentration from a local clock, which can be triggered
by an outer parameter. I f that clock is triggered, a gradient of frequencies
and a pattern of phase differences can easily evolve and function as a timing
parameter in ontogenesis. This concept has been analyzed for the develop­
ment and regeneration in hydra as well as for the problem of positional
projection in early amphibian life ( 173 ) .
7. Oscillations reflect an ev.oltttionary process. Cross-coupling structures
and feedback networks are emerged properties of living structures and in­
herent in all dynamic phenomena in biology. If we observe oscillatory and
highly complex transients of components in intact cellular systems such as
glycolysis of yeast, they can be understood as a result of evolution yielding
kinetic optimization of a pathway. Such a process leads to a dynamic struc­
ture of metabolism, which displays efficient response times and appropriate
damping rates, allowing the controlled system to react in an adapted manner
to continuously changing external conditions.
Oscillatory phenomena have been postulated as an essential part of the
evolutionary process of life, forming the basis of a space differentiation
where gradients of chemical potentials are produced by oscillating chemical
systems ( 166, 167 ) . The existence of either a steady state or a limit cycle
might bring a selective advantage and generate the evolution of the given
regulatory system ( 16 1 ) . Indeed, in a general theory of evolution it has
been pointed out that the very properties outlined above-autocatalyis and
oscillations around steady states-are the tools of evolution, which in an
 254 HESS & BOITEUX

oscillating process continuously select and optimize dynamic structures

( 174 ) . This process will go on as long as growing systems exist.

ACKNOWLEDGMENTS

We thank Drs. H. Degn and E. Chance for stimulating discussions and

suggestions.

DEFINITIONS

Oscillatory kinetics in biochemistry can be described by a number of formal

parameters applied with the following definitions. First of all, oscillatory kinetics
Annu. Rev. Biochem. 1971.40:237-258. Downloaded from www.annualreviews.org

must be distinguished from pseudo-oscillatory phenomena such as those displayed by

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systems in the form of overshoot-undershoot phenomena resulting from a number of

monotonically decaying functions. These phenomena are non periodic-the time be­
tween maxima is not constant and only a finite number of cycles is observed, depend­
ing on the number of functions (for details see 145).

A mplitude and frequency.-As summarized by Higgins (145), true oscillatory

kinetics can generally be described in the following form.

yet) = yet) + A (t)P(t, w)

where y is the mass or concentration of an oscillatory component, yet) the apparent

mean value of y over a short-time average, A (t) the amplitude-modulating function
being variable or constant, and p et, w ) the periodic function with the normalized
amplitude.
In case of more complicated oscillation such as double periodicities, the kinetics
might only be described by a sum of several periodic functions. The variation of the
frequency w as a function of time should be given in radians X min-1 per unit time.

Phase difference.-The relationship of several variables of the nonlinear oscillatory

sy�tem is described by the phase difference (t..), for which a crude average value can
be defined :

t.. =
.1mox +
--- 2-Amin
--

here t1mar. = 27rtm/r gives the phase difference of the maxima of two variables of the
same frequency : r = period, ( ± ) tm = time difference between the maxima of the two
components, t.min is the respective phase differcnce of thc minima. More detailed
analytical data can be obtained by application of the graphic phase plane procedure.

Q value.-The time in 7r X cycies in which the amplitude of the oscillation is re­

duced to lie of its initial value.

Damping factor.-The damping of oscillations can be described by the damping

factor, which is defined as a ratio of the amplitude of two successive deflections of the
trace in the same direction.
 OSCILLATORY PHENOMENA IN BIOCHEMI STRY
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