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Catalase:Anoldenzymewithanew
role?
ArticleinCanadianjournalofbiochemistryandcellbiology=Revuecanadiennede
biochimieetbiologiecellulaire·November1984
DOI:10.1139/o84-129·Source:PubMed
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MairePercy
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Catalase: an old enzyme with a new role?'
MAIREE. PERCY
Department
of Obstetrics and Gynaecology, Mount Sinai Hospital
and the Universio cf Toronto, BOO University Avenue,
Toronto, Ont., Canada M 5 6 1x5
Received October 20, 1983
Percy, M. E. (1984) Catalase: an old enzyme with a new role? Can. J. Biochem. Cell Biol. 6 2 , 1006- 1014
Although animal catalase has been studied for decades, its physiological role has remained perplexing. It has two enzymatic
functions, not only catalyzing the breakdown of H202into O2 and H20, but also in the presence of low concentrations of H202
catalyzing the oxidation of electron donors such as ethanol or phenols. In this article, I have summarized some well-known
properties of the enzyme and have also described several recently discovered features. Of particular interest is the finding that,
although catalase has been regarded as an intracellular enzyme, there is published evidence for its association with the plasma
membrane of the erythrocyte. Moreover, recent work from my laboratory indicates that in vitro at alkaline pH in the presence of
~ g " , the biologically active diphenols (a-3,4-dihydroxyphenylalanine and the P-adrenergic agonists isoproterenol,
norepinephrine, and epinephrine) appeaP to function as electron donor substrates for human erythrocyte catalase and inhibit the
production of O2from H202at micromolar concentrations. The P-adrenergic antagonist propranolol inhibits O2production much
less effectively and appears to competitively inhibit the reaction of catalase with epinephrine. These observations suggest an
analogy between catalase and the B-adrenergic hormone receptor and raise many questions of interest to basic science, health,
and disease.
Percy, M. E. (1984) Catalase: an old enzyme with a new role? Can. J. Biochem. Cell Biol. 62, 1006- 1014
Bien que nsus connaissions la catalase animale depuis des dkcennies, son r61e physiologique est derneurC une cause de
perplexitk. Elle exerce deux fonctions enzymatiques: non seulement elle catalyse la degradation de H202en O2 et H20, mais en
presence de faibles concentrations de H202, elle catalyse aussi l'oxydation de donneurs d.'Clectrons comme 1'Cthanol s u les
phenols. Nous rCsumons ici certaines propriCtCs bien connues de l'enzyme et nous en decrivons aussi quelque autres rkcemment
dCcouvertes. La catalase a toujours kt6 considCree comme une enzyme intracellulaire, mais on a maintenant des preuves de son
association avec la membrane plasmique des krythrocytes. Be plus, nous avons rtcemment montrC dans mon laboratoire qu'in
vitro, h pH alcalin et en presence de MgL', les diphenols (B-3,4-dihydroxyph6nylalanine
et les agonistes P-adrknergiques:
isoprotCrCno1, nsradrknaline et adrknaline) biologiquement actifs semblent agir comme substrats donneurs d'tlectrons pour la
catalase Crythrocytaire humaine et ils inhibent la production de O2 depuis H202 des concentrations micromslaires. La
propranolol, un antagoniste f3-adrknergique, inhibe la production d'02 d'une fason beaucoup moins efficace et inhiberait de
fagon comp5titive la rCaction de la catalase avec l'adrknaline. Ces observations sugg2rent une analogie entre la catalase et le
recepteur de l'homsne f3-adrenergique et soulkvent plusieurs questions interessantes concernant les sciences fondamentales, la
sante et les maladies.
[Traduit par la revue]
Introduction
Animal catalase (JZC1.1 1.1.6; H,o,:H,o,
oxidom
reductase) is one of the most intensively studied of all
mammalian enzymes. In 1818 ~
h observed
~ that
~
hydrogen peroxide was decomposed by animal tissues
with the liberation of gaseous oxygen: ZH,O, +, ZH,O
+ 0,. Eighty-thee years later, in 1901, L~~~ established that this effect was due to a specific enzyme which
he named catalasec 1923, warburg suggested that
catalase was an iron-containing enzyme because it was
inhibited by cyanide. Evidence that its prosthetic group
Was hematin was provided by Zeile and Hellstr~min
1930.
~ Thedenzyme was first crystallized from beef liver
by Sumner and
in
It has since been
crystallized from liver and erythrocytes of a number of
animal species, including the human. It has also been
from bacteria. The primary
beef
liver catalase has been established, but the complete
sequence of human catalase is not yet known. All of the
animal catalases appear to consistof a complex of four
ABBmV1"loNs:
'-P-3,4-dih~drox~~hen~1a1anine;
subunits. Each subunit has an apparent molecular
p1, isoelectric point; AMP, adenosine msnophosphate; ATP,
weight
of about 60 000 and contains one ferriprotoporadenosine triphssphate; GTP, guanosine triphosphate; SDS ,
phyrin
(hematin) group. Upon dilution, the tetramer
sodium dodecyl sulfate.
and monomers with concomitant
l
~ by Canada
~ Health~ adWelfare,
~
grant
~ N ~ . ~dissociates
~ into dimers
d
loss
of
activity.
Although
the enzymatic reactions of
6a-2135-53. h e h s t i h k Mati~ndde la SmG et de la Recherche Medicale'(France), and the Banting Research Foundation. catalase have been intensively studied, the exact mecha1937e
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nism of catalysis has not been established and its
physiological role has remained perplexing. The puzzle
is the following. Firstly, catalase has a very high
turnover number, decomposing H202 into O2 and H20
at an exceedingly high rate (catalatic activity). This
activity is not its only function. In the presence of very
low concentrations of H202
M), it will also
oxidize electron donors such as ethanol or phenols
(peroxidatic activity). Secondly, the level of H202 is
regulated physiologically at concentrations of
lo-' M. At these H202 concentrations, another enzyme, glutathione peroxidase , is believed to decompose
the H202because it has a higher affinity for H202than
does catalase. Thus it has been argued that the main
function of catalase in tissues must be peroxidatic.
Finally, in some cells such as the erythrocyte, the
concentration of suitable electron-donor substrates for
catalase has been estimated to be so low that there have
been doubts that even the peroxidatic activity is of any
consequence ( 1-4).
Because catalase has two enzymatic functions and the
fact that H202 production in different tissues can vary
within a wide range, the actual function of catalase may
vary from tissue to tissue or from one subcellular
compartment to another. Thus it seems logical to
conclude that catalase may react either catalatically or
peroxidatically depending on the microenvironment of
the cell. Cellular reactions leading to the production of
H202 and its catalase-mediated destruction are summarized in Fig. 1.
Tissue distribution and subcellular localization
Catalase is ubiquitously present in aerobic cells
containing a cytochrome system. It is missing in most
anerobic organisms, but occurs in the radioresistant
species Micrococcus radioresistarzs. These observations have prompted speculation about its in vivo role
(1-3).
Catalase is most abundant in liver, kidney, and
erythrocytes and is least abundant in connective tissue.
In the liver cell, catalase is mainly located in peroxisomes, but it has also been identified in the endoplasmic
reticulum and cytoplasm (1-3). Although it was previously believed that catalase was present in mitochondria, this view has been questioned (5). Catalase-rich
peroxisomes have been visualized in myocardium and
smooth muscle fibres. In skeletal muscle, however,
catalase seems to be associated with the sarcoplasmic
reticulum. In muscle it is the aerobic type I fibres which
possess catalase; the anerobic fibres contain very little.
The highest activity has been found in slow oxidative
muscle. In a given muscle type, the catalase activity
varies from one region to another (6,7). Catalase in the
mature red blood cell has been regarded as cytoplasmic.
However, several reports have provided evidence that it
t
H i q reaction
x'
compound I
FIG. 1. Cellular sources of hydrogen peroxide and its
catalase-mediated destruction. ((a) Flavin enzymes (e.g.,
uricase, D-amino acid oxidase). (b) Autooxidation reactions
(e.g., of thiol compounds, ascorbate, hydrazines). bc) Superoxide dismutase. X represents a site on the catalase apoprotein
which is postulated to undergo cyclic oxidation and reduction
during the catalysis. The iron (Fe) is located in the hematin
prosthetic group, represented by L. An hydroxyl ion is
postulated to be linked to the 6th coordination position of the
Fe in compound I, the primary catalase-peroxide complex.
The representation for active sites of catalase and compound I
are hypothetical. (After Aebi and Suter (2) and Chance et al.
is associated with or present in the erythrocyte membrane (8- 11). In liver cells, catalase is synthesized as a
monomer in the endoplasmic reticulum and is rapidly
transferred to the peroxisomes where final assembly to
the tetramer takes place (12).
It is currently believed that both catalase and glutathione peroxidase contribute to the breakdown of H202
in vivoand that both enzymes have important functions,
their relative contribution being dictated by their subcellular distribution, their localized concentration, and
the availability of H202, hydrogen donors, and reduced
glutathione. In liver, the subcellular distributions of
these two enzymes are strikingly different. Glutathione
peroxidase is located mainly in the mitochondria1 matrix
and the cytosol, whereas catalase is highest in the
peroxisomes. Even though erythrocytes contain no
mitochondria, they contain both catalase and glutathione peroxidase, and in these cells glutathione peroxidase is believed to be of major importance in the
breakdown of H202. However, the relative contribution
of catalase should increase with increasing concentration of H202(3-5).
Hydrogen peroxide is a normal cellular metabolite as
well as a potentially reactive form of oxygen which
could cause oxidative damage to tissue. It readily
permeates biological membranes and is believed to
diffuse from one cell compartment to another, into
interstitial fluid, and into the circulation. The intertissue
and intracellular partitioning of catalase and glutathione
peroxidase would appear to provide a dual and complementary defence mechanism against this mobile source
of oxidative stress.
The reader is referred to Refs. 3 and 4 for additional
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1008
CAN. J. BIOCWEM. CELL BIOk. VOL. 62. 1984
information about the roles of glutathione peroxidase,
glutathione S-transferase, and superoxide dismutase in
hydrogen peroxide metabolism.
as
Catalytic reactions
Chance was able to resolve the catalase reaction cycle
into two distinct steps by the study of spectral changes
during catalysis (13, 14). In the first step, the native
ferric hemoprotein (free catalase) reacts with H202 to
form the primary complex: a green, spectrally distinct
form, called compound I. In the second step, two
electrons are transferred from an electron donor to form
water and an oxidized product. The electron donor can
be either a second molecule of H202 (catalatic mode) or
another substance such as methanol, ethanol, or formic
acid (peroxidatic mode). With certain other substrates
(e.g., phenols), two electrons are not transferred simultaneously. The rate of transfer of the second electron is
slow relative to the transfer of the first and results in the
steady-state accumulation of a red, one-electron, reduced form of compound I, which is termed compound
11. Compound I1 is spectrally distinct from free catalase
and from compound I and is an inactive form of the
enzyme. A third catalase-peroxide complex, compound
111, can be identified spectroscopically. This is obtained
by treating compound I1 with H202. The formation of
compound I11 is reversible and, on standing, compound
I1 is slowly reformed (I).
The overall reactions of catalase may be summarized
where AH2 represents a two-electron donor, and
[I]
H202 + AH2 -+ A
+ 2H20
where AH represents a one-electron donor (I 5, 169. In
the catalatic mode, AH2 is a second molecule of H2Q2.
Although the catalatic and peroxidatic reactions are in
many ways well characterized, neither the mechanism
of formation nor the structure of compound I, 11, or 111
are completely understood. Moreover, the exact mechanism of catalysis by catalase has remained elusive.
Nevertheless, the splitting of H2Q2 to H 2 0 and O2 may
be regarded as a special case of the peroxidatic reaction
in which H202 serves both as a substrate and an
acceptor. The peroxidatic reaction is the rule in the
presence of an acceptable electron donor when the H202
concentration is low or when the substate is an alkyl
peroxide. Finally, if a high steady-state level of compound I is generated in low H 2 0 2 concentration or with
alkyl peroxides as substrate in the absence of an electron
donor, the spontaneous decomposition of peroxide will
predominate, presumably via the oxidation of an internal hydrogen donor in the catalase apoprotein (1, 3,
15-17).
The following scheme by Keilin and Nicholls summarizes the known reactions of catalase (18):
catalase
/
D2, D4. D5
compound I1
\
'L
compound I11
Catalase
D6
---+
compound I
D3, D4, D6
catalase
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REVIEW /
where the hydrogen donors have been indicated by Dl to
D6: Dl = ascorbate or ferrocyanide; D2 = phenols; D3
= alcohol or formate; D4 = sodium nitrite; D5 = azide
or hydroxylamine: D6 = hydrogen peroxide. Catalase
from liver will also catalyze the oxidation of NADH and
NADPH by H202;this reaction is enhanced by thyroxine, estradiol- 17, and various phenol compounds.
The regulation of catalase expression and catalase
heterogeneity
Catalase expression in mammals is under complex
genetic control. Studies in the mouse have revealed that
there is only one structural gene for catalase. Mouse
catalase is a glycoprotein and five distinct electrophoretic forms differing in sialic acid content are
differentially distributed among different tissues and
subcellular organelles. The rate of breakdown of catalase in the liver is controlled by a regulator gene that is
unlinked to the structural gene. A different regulator
gene is believed to control catalase turnover in the
erythrocyte (19,20). In addition to these complexities, it
is now believed that enzymatically active catalase
purified from liver may be a proteolytic cleavage
product of a larger precursor form (21). It has been
suggested that the cleavable peptide may play a role in
the transport of the enzyme across cellular membranes
or may occupy a significant niche in the sequence of in
vitro degradation.
These studies of catalase in the mouse may serve as a
model system for unravelling the complexities of catalase expression in man. The structural gene for human
catalase is on chromosome 11, but tissue-specific
regulator genes have not yet been mapped. Putative allelic
variants of the human structural gene for catalase have
been described. The first structural gene mutation
reported for human catalase was observed by Takahara
for Japanese individuals who exhibited a hereditary
deficiency of blood catalase. This rare autosomal recessive disorder was termed acatalasemia (22). The structural defect leads to a decrease of catalatic activity early
in the life of the erythrocyte to about 1% of that in the
control. It is now clear that acatalasemia must be
considered to be a group of mutations which all lead to a
change in the activity level of the normal enzyme. In
many but not all instances the deficiency of erythrocyte
catalase is combined with a deficiency of tissue catalase.
In some cases, the enzyme variant is characterized by
poor stability; in others, a catalase variant of low activity
is synthesized. Surprisingly, individuals with acatalasemia are not severely affected, perhaps because the
deficiency is never complete and seems to be compensated by an increased activity of the glutathioneregenerating system, notably the hexose monophosphate shunt (2, 23-25).
Feinstein and colleagues (26, 27) have described a
series of five radiation-induced mutants for erythrocyte
catalase in the mouse. One of these is acatalasemia; the
other four are hypocatalasemias. Genetic analysis has
shown that this group of erythrocyte mutants is controlled by five different alleles at a single locus corresponding to the site of the catalase structural gene. The
suggestion has been made that the acatalasemic mutant
is a structural mutation that affects the enzymatically
active site of the molecule, while not altering the
immunospecificity of the protein. Two additional hypocatalasemic mouse mutants have been subsequently
described ( 19).
In addition to the human acatalasemic variants,
several rare phenotypes of catalase have been identified
by electrophoresis in population surveys in North and
South America. They have been attributed to the
heterozygosity of different mutant alleles (28, 29).
Distinct from the electrophoretic variation associated
with variant structural alleles of human catalase is a
heterogeneity which seems to be induced or increased by
storage (30). Morikofer-Zwez et al. have suggested that
this heterogeneity is the result of artifactual oxidation of
unessential sulfhydryl groups which cause the introduction of net negative charges in the molecule (31).
However, Bonaventura et al. have pointed out that the
heterogeneity which this group has defined may be
distinct from that seen in freshly isolated erythrocytes
(32). One possible source of heterogeneity in freshly
prepared tissue extracts or in purified catalase that has
been consistently overlooked is the interconvertibility of
free catalase and compounds I, 11, and 111. Catalase has
been shown to exist in-the form of compound 1 in tissues.
As evident from the scheme of Keilin and Nicholls (18),
these interconversions can be chemically manipulated,
and it should be feasible to determine whether or not the
electrophoretic mobility or the isoelectric focusing
pattern of free catalase differs from that of compound I,
11, or 111.
It is clear from the preceding discussion that the
activity of catalase is regulated at several different
levels: by the structural gene, by genes which epigenetically modify and (or) regulate the turnover of the
catalase protein, and by the interaction of catalase with
its substrates. Abnormalities at any of these levels might
affect the activity of catalase.
The relation of catalase and the diphenoloxidases
Although the reactivity of catalase with electron
donors such as ethanol has been thoroughly studied, there
has been little thought about the potential reactivity of
animal catalase towards biologically active phenols such
as DOPA and epinephrine ( 17). The ability of catalase to
oxidize epinephrine has long been recognized by microbiologists, however, and this property is exploited
routinely to identify catalase-positive microorganisms.
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1010
CAN. J. BIOCHEM. CELL BIQL. VQL. 62. 1984
Enzymes which catalyze the oxidation of DOPA to
melanin or epinephrine to adrenochrome have been
described in a variety of mammalian tissues by many
different groups, but the possibility has never been
considered that these enzymes may be catalases (3342). Because the reaction conditions for studying these
enzymes have been so diverse, it is also difficult to say
whether or not the catalytic activity in question has been
the same in all of the studies. Nevertheless, the activities
which have been described do not catalyze the oxidation
of tyrosine to melanin and thus appear to be distinct from
the enzymes which have been termed "tyrosinases" (43,
44). Moreover, Inchiosa et al. showed that the
epinephrine-oxidizing enzyme in their study was distinctive from cytochrome c oxidase and ceruloplasmin
(37).
Demos, Tuil, and colleagues have focused on watersoluble, ~g'+-dependent enzymes which have been
called "diphenoloxidases" (39-42, 45-48). Diphenoloxidase activity in cell extracts is revealed at pH 8.7- 18
in two ways: (a') spectrophotometr4cally with epinephrine as substrate by following the absorbance increase at
485 nm (i.e., the conversion of epinephrine to adrenochrome which has an absorption maximum at 485 nm);
(is') visually, after resolution by gel electrophoresis or
isoelectric focusing, by immersion of the gel in the
presence of air and light in a solution of DOPA. In the
first assay the substrate is first incubated in the presence
of air and light for 30 min; upon addition of the enzyme
and a catalytic amount of adrenochrome, epinephrine
oxidation occurs very rapidly without a lag period. In the
latter case, a diffuse deposit of melanin f o m s over the
enzyme bands.
In rabbit, rat, mouse, and chicken, the diphenoloxidases are found in highest concentration in kidney,
erythrocytes, and platelets, and their isoelectric focusing pattern is species and tissue specific (45-48). In
man, diphenoloxidase activity is high in platelets and
erythrocytes. Of particular interest is the fact that in
erythrocytes, at least, the enzyme activity is membrane
associated as well as cytoplasmic and dependent on
~g~~ ions (45-48). The isoelectric focusing patterns of
human platelet and erythrocyte diphenoloxidase are not
identical. The PI'S of the platelet bands are 6.65 and
6.15, whereas the p%'sof the red cell bands are 6.8 and
6.7. Diphenoloxidase activity is inhibited by
M
KCN (partial),
A4 lead acetate (complete), digestion with pronase for 18 min at 3T°C (complete), and
heating for 10 min at 65°C (complete) (45, 46).
Demos began studies of the diphenoloxidases because
he felt that an abnormality in catecholamine metabolism
might be a cause of the microcirculatory disturbances
which ape associated with the progressive muscular
dystrophies (49). His studies have revealed that platelet
diphenoloxidase is significantly reduced in patients with
all four major forms of progressive muscular dystrophy
(Becker and Duchenne types, X-linked recessive; limbgirdle, autosomal recessive; fascioscapulohumeral,
autosomal dominant), as well as in many carriers of
these disorders (40). The reduction in platelet diphenoloxidase activity is greatest in the X-linked lethal
recessive Duchenne form and is fourfold lower in
affected patients than in controls.
I have confirmed Demos' observations that platelet
diphenoloxidase is 50% reduced in known carriers of
Duchenne muscular dystrophy relative to sex- and
age-matched controls (unpublished observations) and
have extended Demos' studies of these diphenoloxidase
enzymes. While screening a number of catecholaminemetabolizing enzymes for diphenoloxidase activity,
crystalline beef liver catalase and catalase purified to
95% homogeneity from human erythrocytes isolated in
1% disodium EDTA were found to have diphenoloxidase activity as assessed by the two assay methods of
Demos (41). Moreover, when the catalases were resolved by isoelectric focusing on polyacrylamide and
duplicate sections of the gel were analyzed in parallel,
the banding patterns obtained with stains for catalase
activity and diphenoloxidase activity were superimposable. The addition of 1.5 mM H202 to the solution for
revealing diphenoloxidase activity reduced the necessary staining time from a couple of hours to a few
minutes, but did not alter the banding pattern of the
enzyme activity. Superimposable isoelectric focusing
patterns of catalase activity and diphenoloxidase activity
were also observed in freshly prepared extracts of
erythrocytes and platelets, although the patterns were
not identical in the two tissues. Moreover, antiserum to
catalase has been shown to remove both catalase and
diphenoloxidase activity from platelet and red cell
extracts. These observations have suggested that catalase and diphenoloxidase may be one and the same
enzyme (unpublished observations).
Intuitively, there are several possible explanations for
the reduced diphenoloxidase activity in the progressive
muscular dystrophies, and the abnormalities which
Demos has described might well be a secondary effect
associated with these disorders (50). However, the
suggestion that diphenoloxidase and catalase might be
the same enzyme raises the possibility that different
abnormalities in genes which epigenetically modify
catalase or which regulate its turnover might be a
fundamental problem in these disorders.
Activity of catalase with P-adrenergic agents and a
possible analogy with the P-adrenergic hormone
receptor
Using Demos' gel method for visualizing enzyme
activity with the addition of 1.5 m M H202 to the
developing stain, I established that purified beef liver
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catalase and purified human red cell catalase oxidized
not only DOPA and epinephrine, but isoproterenol,
norepinephrine, and propranolal as well. Isoproterenol,
norepinephrine, and epinephrine are (3-adrenergic agonists; propranolol is a P-adrenergic antagonist and a
commonly used antihypertensive agent. To establish the
relative activity of catalase with these substrates, I tested
the ability of these substrates to inhibit the catalatic
production of O2 from H202 in the Rank oxygen
electrode in Tris-citrate buffer (pH 8.7) containing
4 mM MgW . The rationale for this approach is based on
the knowledge that, in the presence of low concentrations of H202, suitable electron-donor substrates will
partition the catalatic activity of catalase into the
peroxidatic mode (formation of compound I1 and subsequent substrate oxidation) with concomitant inhibition
of 0 2 production from the H202. In these experiments
the concentration of H202was fixed at 1.5 mM and the
concentrations of the second substrates ranged from 0 to
3X
M. L-DOPA and all three agonists were found
to inhibit O2 production from H202 in an apparently
competitive manner even at concentrations of lw4lo-'M. Propranolol inhibited O2 production as well,
but less effectively than any of the other substrates
tested. It also appeared to competitively inhibit the
reaction of epinephrine with catalase as judged by the
reaction kinetics. The relative affinities for the substrates tested were established to be L-DOPA > isoproterenol > norepinephrine > epinephrine >>> propranolol, the apparent &'s ranging from 0.032 mM for
L-DOPA to 1.7 mlW for D,L-propranolol (unpublished
observations). The structures of the compounds tested in
these experiments are given in Fig. 2. It has been
previously noted that the three open-chain agonists have
the ability to oxidatively cyclize (38).
Although this suggestion must be regarded as preliminary, the finding that catalase will react with
P-adrenergic agonists with surprisingly high affinity
forces one to wonder if there is an analogy between these
in vitro reactions and the reactions of the P-adrenergic
hormone receptors with P-adrenergic agonists and
antagonists in cells.
Many different cells respond to the catecholamine
hormones epinephrine and norepinephrine. One of the
ways these hormones exert their effects is by binding to
the hormone receptors of these cells. The binding to the
hormone receptor results in activation of the catalytic
component of adenylate cyclase and the generation of
cyclic AMP (5 1). A schematic relationship between the
hormone receptor and other components of the adenylate cyclase holoenzyme system is shown in Fig. 3.
The p-adrenergic hormone receptor has been difficult
to characterize, as the properties of isolated receptors are
not identical to those in membrane fractions or whole
cells (52). Nevertheless, binding characteristics of the
+ + - l;- + -
OHH
H
H
- H
H
C H ~ isoprotereno~
CH3
HO
i
aQQnists
norepinephrine
H0
epinephrine
HO
H OHH H H
I
I
I
I
I
o-c-C-C-N-C-CH~
I
I
H H
alprenolol
Cl-$
!-?=
?-H
H H
H
I
I
1
antagonists
H OHH H H
l
l
0-C-6-C-N-C-CH~
propranotol
I
FIG.2. A comparison of the chemical structures of DOPA,
some P-adrenergic agonists, and some P-adrenergic antagonists.
P-adrenergic agonists to these have been defined and it
has been established that isoproterenol is a more potent
agonist than epinephrine or- norepinephrine (53j. The
antagonists, of which an example is propranoloI, bind to
the receptor but do not stimulate adenylate cyclase
activity and competitively inhibit agonist binding to the
receptor. Persistent exposure of cells to P-agonists
leads to a decreased responsiveness to subsequent
catecholamine stimulation. h4g2+ promotes binding of
agonists, but not antagonists, to the receptor (52). In the
frog erythrocyte, the purified hormone receptor has an
apparent molecular weight of 58 000 as estimated by
SDS-polyacrylamide gel electrophoresis (54, 55).
From Fig. 2, it is evident that the agonists and
antagonists have side chains that are chemically similar,
but that their aromatic components are not. The steric
arrangement of functional groups in the agonists has
been hypothesized to play an important role in the
production of their biological effect. Conformational
analysis of various P2-adrenoceptor stimulating agents
has suggested that the crucial feature may be the
stereochemical arrangement of their two catechol hydroxyls and the hydroxyl and N groups of their alkyl side
chains (56). The reactions of catalase with the Padrenergic agonists draw attention to an additional
property: namely their ability to function as electron
(hydrogen) donor substrates for catalase.
CAN. J. BIOCHEM. CELL BIOL. VOL. 62, 1984
TABLEI . Similarities between catalase and the P-adrenergic hormone receptor
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CAT
R
MW of basic subunit
58 000 (human red cells)
58 000 (frog red cells) (54)
I, N, and E (beta agonists)
Inhibit the catalatic reaction of
CAT; relative inhibition:
I>N>E*
Propranolol (beta antagonist)
Inhibits the reaction of CAT with
I, N, and E*
Binds to R and indirectly stinmulates adenylate cyclase; relative
binding affinity: I > E, and N
(53)
Binds to R, but does not stimulate
adenylate cyclase; inhibits
agonisa binding to W 653)
Promotes the reaction of CAT
with I, N, and E*
Promotes the binding of agonists
to W (51)
Inhibits CAT by binding to
henlatin (61)
Indirectly stimulates adenylate
cyclase ( 5 1)
NOTE:CAT, catalase; R. p-adrmergic hormone receptor; MW. molecular weight; I. isoproterenol; N,norepinephrine; E, epinephrine.
*In i~itroat alkaline pH in the presence of Mg2+(unpublished results). The concept that there is a membrane-bound fc)rni of catalase
which interacts with p-adrenerpic agents in viva is speculative. However, several reports have suggested that catalase is associated with
or present in the erythrocyte membrane (8- 11). The enzyme diphenoloxidase, which may be related tocatalase. has also been identified
in the erythrocyte rnelnbrane (45-48).
Cyclic AMP
FIG.3. Schematic representation of the relationship between
the P-adrenergic homone receptor and other components of
the adenylate cyclase holoenzyme responsible for regulation
by hormones and GTP. The homone receptor (R) is visualized
as spanning the plasma membrane and having different
domains which have functions for attachment to the membrane
and linkage with a nucleotide regulatory unit (N) that binds
GTP. The N unit forms a bridge between W and the catalytic
component of adenylate cyclase (C) at the internal face of the
membrane. (After Rodbell (51).)
Because the P-adrenergic agents react with catalase in
vitrs and bind to the P-adrenergic hormone receptor in
vivo, it is tempting to speculate that the P-adrenergic
homone receptor might be a membrane-associated form
of catalase. Thus, the steseochemical conformation of
the alkyl side chains of P-adrenoceptor stimulating
agents might direct their binding to specific sites in the
hormone receptor and the electron-donating properties of
the two catechol hydroxyls might be essential for theis
biological activity.
Certain properties of catalase and the hormone recep-
tor are compared in Table 1. Attention is drawn to the
similarity in size of their basic subunit; their reactivity
with isoproterenol , norepinephrine, and epinephrine;
the role of propranolol as an inhibitor; and the ability
of hIg2+to promote reactions with the agonists. For
interest, the effect of fluoride on the activity of catalase
and the adenylate cyclase holoenzyme has also been
included.
Although the reactions of catalase with the Padrenergic agents must currently be regarded as an in
vitro novelty, this reactivity merits further investigation.
The hypotheses that catalase and the diphenoloxidases
are structurally related and that the P-adrenesgic hormone receptor is a membrane-associated form of catalase can be tested. Even if these putative identities
should be disproven, it is nevertheleis essential to
determine whether P-adrenergic agents will react with
catalase in intact cells at concentrations used to stimulate
adenylate cyclase activity. In addition, given that
catalase is associated with the plasma membrane in at
least some cells, the effect of ethanol, a known catalase
substrate, on the activity of adenylate cyclase should not
al
have been devised
be overlooked. ~ x ~ e r i m e n tsystems
to measure compound I and 11 formation and catalase
inhibition in intact cells and perfused organs (15, 57,
58). It will clearly be of interest to study the effects of
biologically relevant electron donors such as ethanol and
the P-adrenergic agents on catalase using intact cells and
organs and to correlate the results with effects on the
adenylate cyclase holoenzyme system. That such investigations might prove to be rewarding is already
implied from the work of Jones et al. (159, who have
shown that a-methyl DOPA, a commonly used anti-
Can. J. Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by University of Toronto on 10/15/15
For personal use only.
hypertensive agent, inhibits the catalatic activity of Dr. and Mme Demos, Dr. and Mme Tuil, and other staff
catalase in isolated hepatocytes owing to the drug- of INSERM U 193 for their generous hospitality.
induced formation of compound I1 and the presumptive
oxidation of a-methyl DOPA via the peroxidatic path1. Deisseroth, A . & Bounce, A. L. (1970)Playsiol. Rev. 50,
way (15, 59). Jones et al. have suggested that this
319-375
reaction of catalase with a-methyl DOPA may contribute to the known toxic side effects which have been 2. Aebi, H. & Suter, H. (1971) Adv. Hum. Genet. 2,
143- 199
associated with the drug (15). However, it is also possible that the reactivity of catalase with a-methyl 3. Chance, B., Sies, H. & Boveris, A. (1979)Physiol. Rev.
59,527-6Q5
DOPA accounts for the antihypertensive effect of the 4. Sinet, P. M. (1982) Ann. N.Y. Acud. Sci. 396, 94-107
drug.
5. Joncs, D. P., Eklow, L., Thor, H. & Orrenius, S. (1981)
Arch. Biochem. Biophys. 210, 505-516
6. Christie, K. N. & Stoward, P. J. (1979) J. Histochem.
Conclusions
Cytochem. 27, 814-819
In 1938, J. B. S. Haldane stated in his contribution to
7. Jenkins, R. R. & Tengi, J . (1981) Experientia 37,67-68
the monograph Perspectives in Biodzemistry: "The cells
8. Allen, D. W., Cadman, S. & McCann, S. R. (1977)
of the body are very largely autonomous. So far as we
Blood49, 113-123
know they all make their own enzymes. Catalase is
9. Snyder, L. M., Liu, S. C., Palek, J., Rulat, P. &
probably just as important as insulin. We do not know
Edelstein, L. (1977) Biochim. Biophys. Acta 470, 290what a man, or a rat, without catalase or even with too
302
little catalase, would be like. And since we cannot stop 10. Deas, J. E., Lee, L. T. & Howe, C. (1978) Biochem.
Biophys. Res. Commun. 82, 296-304
the cells from making catalase . .. we are not likely to
11. Aviram, I. & Shaklai, N. (1981) Arch. Biochem.
know for a considerable time" (2, 68).
Biophys. 212, 329-337
Studies of acatalasemia in man and mouse have
implied that even a substantial reduction in the catulatic 12. Lazarow, P. B. & de Duve, C. (1973) J. Cell Biol. 59.
507-524
activity of catalase has minor physiological signifi- 13. Chance, B. (1947) Acta Chem, Scand. 1, 236-267
cance. However, the question of whether the acatala- 14. Chance, B. (1952) Arch. Biochem. Biophys. 41, 404semic mutations also affect the peroxidatic activities of
414
catalase with DOPA or the P-adrenergic agents has not 15. Jones, D. P., Meyer, D. B., Andersson, B . & Orrenius,
S. (1981) Mol. Pharmacol. 20, 159-164
been examined. Because of this gap in our knowledge
and because of the possibility that the peroxidatic 16. Jones, P. & Bunford, H. B. (1977) J. Theor. Biol. 69,
457-470
activity of catalase is physi~logicallymore significant
than its catalatic activity, Haldane's philosophical com- 17. Oshino, N., Oshino, R. & Chance, B. (1973) Biochem.
J. 131, 555-567
mentary still applies.
18. Keilin, D. & Nicholls, P. (1958) Biochim. Biophys. Aetu
It is clear from the preceding considerations that
29, 302-307
although catalase may be an old enzyme, it may have 19. Hoffman, H. A. & Grieshaber, C. K. (1974) J. Hered.
some new physiological roles. The evidence that cata65,227-279
lase is associated with or possibly present in the plasma 20. Holrnes, R. S. & Masters, C. J. (1978) Biochem. Genet.
membrane of at least some cells and its demonstrated
16, 171-190
ability to react with biologically significant electron 21. Crane, D., Holrnes, R. & Masters, C. (1982) Biochem.
Biophys. Wes. Commun. 104, 1567- 1572
donors such as ethanol, DOPA, a-methyl DOPA,
norepinephrine, epinephrine, and propranolol raise 22. Takahara, S. (1952) Lancet 2, 1101-1102
many questions for investigation that might have wide- 23. Aebi, H. & Wyss, S. R. (1981)Acta Biol. Msd. Ger. 40,
537-54 1
spread application in the basic science, clinical, and
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