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Isozymes and Heteroenzymes.

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ANGEWANDTE CHEMIE
ion
VOLUME1
*
NUMBER4
A P R I L 1962
PAGE 169-224
Isozymes and Heteroenzymes
BY PROF. DR. T. WIELAND AND PROF.DR. G. PFLEIDERER
INSTITUT FOR ORGANISCHE CHEiMIF, UND BIOCHEMEEHE ABTEILUNG
UNIVERSITAT FRANKFURT AM MAIN
“Heteroenzyme” is the term applied to proteins of different origin that differ in their
physical, chemical, and biochemical properties, but have the same biological action.
Enzymes that have the same origin, and consist of very similar, but distinguishable
proteins have been named “isozymes” [*I; they m y also be designated as “multiple
forms.”
Introduction
As a result of the surprising discovery made in the
thirties that all the important coenzymes have the
same structure regardless of biological origin, biochemists have come to feel that apoenzymes also are
identical in structure. Thus, most have continued to
speak of “an enzyme” in terms of a well-defined molecular species, as for example in the case of pepsin or
catalase. Implicitly, it has been assumed that similar
enzymatic properties are due to a similar, though perhaps not identical, structure.
As will be shown in the examples to follow, it has been
demonstrated a number of times in recent years that
enzymes of similar action, for which the terms “isodynamic” or “homotropic” have been proposed, may
differ from one another either chemically or physically,
depending on the type of tissue from which they originate. We shall designate such enzymes here as heteroenzymes. At the same time indications are increasing
that an enzyme of the same origin may be heterogeneous,
i.e. that it may consist of different molecular species;
these we shall here designate as “multiple forms.”
Heteroenzymes
Even today most biochemical handbooks list the important data concerning each enzyme under one category, regardless of its origin. Yet for many years now
observations have been made, and attention been called
to the fact, that enzymes of similar action but of differ[*] An International Commission on nomenclature is now
engaged in discussing this concept.
Angew. Chem. internat. Edit. / Vol. I (1962) / No. 4
ent origins may be different. Thus, E. Fischer, who was
probably the first to study the specific action of glycosidases, made the following comment in a footnote
written at the end of the last century: “. . . the various
maltases that undoubtedly exist should be termed corn,
yeast, etc. maltases, depending on their origin” [l].
Nevertheless it was 50 years before 0. Warburg was
able to prove that yeast zymohexase (aldolase) differs
from crystalline aldolase of animal origin. Only the
former requires heavy metal ions as cofactor [2]. A
similarly clear distinction was shown to exist between
crystalline alcohol dehydrogenase isolated from horse
liver [3] and from yeast [4]. In these instances the properties of enzymes were compared, even though they had
been isolated from cells of very different origin. On the
other hand, a chemical and biochemicalstudy of enzymes
with the same action isolated from the same organ of
different species is of particular interest. In this connection attention is called to the fundamental studies
of amylases conducted by K. H. Meyer and his coworkers [5-111.
[l] E. Fischer, Ber. dtsch. chem. Ges. 28, 1429 (1895).
[2] 0. Warburg and W.Christian, Biochem. Z. 314, 149 (1943).
[3] R. K . Bonnichsen, Acta chem. scand. 4, 715 (1950).
141 E. Racker, J. biol. Chemistry 184,313 (1950); E. Negelein and
H. J. Wullp, Biochem. Z. 293, 351 (1937).
[5] K . H . Meyer, Ed. H. Fischer, and P. Bernfeld, Helv. chim. Acta
30, 64 (1947); Experientia 3, 106 (1947).
[a] K. H. Meyer, Ed. H. Fischer, P. Bernfeld, and A. Staub, Experientia 3, 455 (1947).
[7] K.H. Meyer, M. Fuld, and P. Bernfeld,Experientia3,411(1947).
[8] P . BernfeZd and H. Studer-Pecha, Helv. chim. Acta 30, 1904
(1947).
[9] P . Bernfeld and M. Fuld, Helv. chim. Acta 31, 1420 (1948).
[lo] P. Bernfeld and M. Fuld, Helv. chim. Acta 31, 1423 (1948).
[ll] P. Bernfeld, F. Duckert, and Ed. H. Fischer, Helv. chim. Acta
33, 1064 (1950).
169
These authors found that crystalline u-amylase isolated
from human saliva is identical with that isolated from
the human pancreas, but differs significantly from that
isolated from hog pancreas. These differencesinvolve
both physical and biochemical characteristics (cf.
Table 1).
LDH species. These components were designated as A
and C (Fig. 1). Shortly thereafter Krebs [181reported that
yeast contains four proteins, each with its characteristic
migration when subjected to electrophoresis and all four
I
Specific activity
pH optimum
Region of stability
Solubility in H20 at pH 8.0
Electrophoretic mobility
at pH 10.14 [cm~/sec.l
Human
u-amy1ase
Hog amylase
lo00
6.9
pH 4.5-11
0.5 %
630
6.9
pH 7-8.5
4%
3.75 x 10-5
3.55 x 10-5
C
A
A
I
Fig. 1. Schlieren diagram: crystalline LDH from bed heart following
electrophoretic separation. Phosphate buffer (pH 5.7); ionic strength:
0.1 ; descending.
From J. B. Neilands, Science (Washington) I I S , 144 (1952).
of which exhibit glyceraldehyde phosphate dehydrogenase activity. Fractional precipitation with nucleic
acid allowed fairly complete separation of these four
proteins. Using starch block electrophoresis Veseil and
Bearn [19] separated human serum and demonstrated
LDH activity in three separate components. At the
same time the authors of the present review initiated a
study “on the differences of lactate dehydrogenases”
[20], previous electrophoretic studies having revealed
several proteins with LDH activity derived from a
variety of organ extracts [21].
By now the number of reports like this has grown to a
point where a special conference held under the auspices
of the New York Academy of Sciences was organized
to deal with the subject of multiple forms of enzymes
1221 and.the question of nomenclature has become real.
The term “isozymes” has been proposed [23] for enzymes that have the same action, i.e., are isodynamic,
and are derived from the same organ, but have different
physical and chemical properties.
Differentiation and Separation of Isozymes
and Heteroenzymes
Multiple Forms of an E w e (Isozymes)
Whereas the description of an enzyme type is complicated by the occurrence of heteroenzymes, it becomes
considerably more so as a result of the discovery that
an enzyme of a single origin may occur in multiple
forms. The first definite observation of this type was
made by Neilands [16] who demonstrated that a previously reported protein component [17] of crystalline beef
heart lactate dehydrogenase [*I constituted a second
[12]R. K. Bonnichsen, Arch. Biochem. Biophysics 12,83 (1947).
[13] 0.Warburg: Wasserstoffiibertragende Fermente. Verlag Dr.
Werner Stinger G.m.b.H., Berlin 1948, p. 54.
[14]F. Kubowitr and P. Off, Biochem. Z. 314, 94 (1934).
[15]G. Pfleiderer and D. Jeckel, Biochem. Z. 329,371 (1957).
[16]J. B. Neilands, Science (Washington) 115, 143 (1952); J.
biol. Chemistry 199, 373 (1952).
[17]A. Meisfer, J. biol. Chemistry 184, 117 (1950).
[*I The abbreviation used in this paper is LDH.
170
Enzymes may be characterized by biochemical,physical,
chemical, and immunological methods. The enzyme
should be available in maximum purity, preferably in
crystalline form.
Biochemical criteria are as follows: (a) turnover number, 1. e.
the number of moles of substrate that react per mole enzyme
per minute; (b) optimum pH and (c) substrate and coenzyme
affinity, expressed by the Michaelis constant, Km,i.e. the
concentration of substrate in moles/liter required for half
saturation of the enzyme. Other criteria include the temperature coefficient and the extent to which various substances
can interfere with the reaction. Also of importance is the use of
[18]E. G. Krebs, J. biol. Chemistry ZOO, 471 (1953).
[19]F. S.Veselland A . G. Bearn, Proc. SOC.exp. Biol. Med. 94,96
(1957).
[ZO] Th. Wieland and G. Pfleiderer, Biochem. Z. 329, 112 (1957).
[21] Th. Wielundand G. Ppeiderer, Angew. Chem. 69,199(1957).
[22] Report, Angew. Chem. 73, 246 (1961);Ann. N.Y.Acad.
Sci. 94, 655 (1961).
[23] C. L. Markert and Molter, Proc. nat. Acad. Sci. USA 45,
753 (1959).
Angew. Chem. internat. Edit. 1 Vol. I 119621 1 No. 4
modified coenzymes, as employed by Kaplan, for example, in
his study of DPN-dependent dehydrogenases [*I. He deterp ~ in a reaction catalyzed by
mined the ratio of v ~ :wpN(ana)
the apoenzyme, when its coenzyme is either DPN or a DPN
analog (abbreviated as DPN(ana)). If this ratio is the same for
two apoenzymes, they are very probably identical. Differences
in this ratio, on the other hand, indicate differences in the
apoenzyme structure [%I.
The following physical methods are available: determination
of molecular weight by the ultracentrifuge; light scattering :
diffusion or osmometry; determination of solubility and
preparation of a salting-out diagram; ultraviolet absorption;
potentiometric titration of acidic and basic groups; determination of the isoelectric point and of the electrophoretic
mobility; rate of migration on ion-exchange columns or uncharged adsorbers such as calcium phosphate; rotational
dispersion ; X-ray crystallography.
Chemical determinations can be carried out and data may
be obtained concerning: terminal amino groups; functional
groups (NH2, OH, SS, SH,etc.); type and number of component groups (e. g . amino acids, coenzyme, heavy metals);
primary structure (sequential analysis) ;oligopeptide mixtures
obtained by proteolysis followed by two-dimensional paper
chromatography and/or electrophoresis (finger-print technique) [25,26].
Immunological tests are particularly sensitive for differentiating or identifying two proteins. In principle, an animal is
sensitized with one of the enzymes and its serum antibodies
are allowed to react with the other enzyme under study. If the
two enzymes are identical, they must require the same amount
of anti-serum for inactivation; the enzymes differ if quantitative differences arise in this test or, occasionally, if one
enzyme does not give rise to any antigen-antibody reaction.
As is true of all biological tests, immunological techniques
are not as quantitative and reproducible with respect to the
detecting of minor differences as generally wished for.
on the method employed for chromatographic separation on DEAE cellulose. In the presence of glucose,
trypsin converts enzyme A into enzyme B. A similar
increase in the proportion of B with respect to A can
be obtained by extracting yeast for 24 instead of the
usual 3 hours. Nevertheless, there is a tendency to label
components A and B as isozymes of hexokinase, even
though upon further careful chromatography, they can
be split into even more components.
Difficulties also arise when an attempt is made to apply
the terms “multiple form” or “heteroenzyme” to
enzymes with a wide region of specificity. For example,
esterases from a single organ that can be differentiated
according to the criteria enumerated above, may still
be termed multiple forms so long as they are tested by
means of a universal substrate. Only detailed consideration of the specificity leads to the recognition that the
enzymes involved have different functions, e.g. that
one is a lipase, the other an acetylcholine esterase, etc.
The same is true for proteinases and most phosphatases.
It is, therefore, only possible to speak of different forms
of an enzyme with certainty when its physiological role
and specificity are known thoroughly. The specificity
with respect to the coenzyme must also be included. For
this reason, for example, the two liver isocitric acid
dehydrogenases are not considered as multiple forms,
since one requires DPN, whereas the other requires
TPN [29].
Differences between Heteroenzymes
Questions of Definition
When multiple enzyme forms are being studied there
is the constant danger that artifacts may result in the
course of tissue preparation and purification. Thus, it
proved possible to decompose animal cytochrome c,
which is a relatively low-molecular weight protein, into
several components of varying specific activity by passing
it through chromatographic columns containing cationic
exchangers. On the other hand, the uniform, purified
enzymes could be transformed into a mixture of multiple
forms by the use of denaturing reagents. As a result,
the occurrence of multiple forms was traced to the
extraction with aqueous trichloroacetic acid in the
course of the preparation of cytochrome [27]. Such artifacts cannot be included in the term “isozyme.”
But where, then, is the difference between artifact and
natural product? According to Trayser and Colowick
[28] yeast contains several hexokinases. These can be
prepared in two crystalline groups, A and B, depending
[*IThe abbreviations DPN and DPNH refer to diphosphopyridine nucleotide, and the reduced form, respectively.
(241 N . 0. Kaplan, M . M . Ciotti, M . Hamolsky, and R . E. Bieber,
Science (Washington) 131, 392 (1960).
[25] Th. WieIand, Angew. Chem. 71, 417 (1959).
[26]H. Tuppy, Naturwissenschaften 46, 35 (1959).
[27]E. Margoliash and J. Lustgarten, Ann. N.Y. Acad. Sci. 94,
731 (1961).
[28]K . A. Trayser and S. P. Collowlck, Arch. Biochem. Biophysics 94, 177 (1961).
Angew. Chem. internat. Edit. 1 Vol. 1 (1962) 1 No. 4
Inasmuch as, in principle, all organisms are capable of
deriving energy in the same fashion (glycolysis, citric
acid cycle), it is reasonable to suppose that each cell has
at its disposal similarly acting enzymes. It is not surprising, therefore, that heteroenzyrnes exhibit marked
differences in their amino acid composition and also
in other properties. An example is the phosphoglucose
isomerase obtained from yeast and the bovine mammary
gland [30], the molecular weight of the former being
145000, in contrast to a molecular weight of 48000 for
the latter.
On the other hand, it seems surprising that heteroenzymes of very different origin exhibit similar properties. For example, the amino acid composition of
glyceraldehyde phosphate dehydrogenase obtained
from yeast and mammalian muscle is quite similar [31].
Chemical differences in the a-amylases derived from
Aspergillus oryzue, 3. subtilis, pig pancreas and human
saliva have been studied by Stein, Junge, and Fischer [32].
Even though all four enzymes have approximately the
same molecular weight (SOOOO), the same prosthetic
[29]A. Kornberg and W. Pricer, J. biol. Chemistry 189, 123
(1951).
[30]A. Baich, R. G. Wove, and F. J. Reithel, J. biol. Chemistry
235, 3130 (1960).
[31]S.F. Velick and S. Uderlfriend, J. biol. Chemistry 203, 575
[19531.
[32] E. A. Stein, J. M.Junge, and E. €
Fischer,
I
. J. biol. Chemistry 235, 371 (1960).
171
group (calcium ion) and a similar turnover number,
their primary structures are quite diflerent. The chemical
difference between enzymes derived from man and hog
is as great as that between enzymes from B. subtilis
and A . oryzue (see Table 2).
i s replaced by a lysine molecule in hemoglobin C, and
by valine in hemoglobin S [37,381.
Similarly, beef, sheep, and pig trypsiw, which are quite
similar in many respects, differ from one another only
in their electrophoretic behavior and in their stability
in alkaline medium. At pH 4.8 the pig enzyme migrates
Table 2. Composition of a-amylasas of different origin. (The numbers
indicato the numbor of amino acid molecules por IOOOOO g.protein.)
I
a-Amylase from
ASpa?Et&
oryzae
Lysino
Ardnino
Wttidine
Bacillus
subtilis
Human
Hog
pancreas saliva
33
11
12
51
35
25
34
33
25
43
50
21
I74
99
197
103
180
94
210
96
66
I7
15
48
58
14
25
31
58
-
60
17
19
89
50
36
91
+
h p u t i c acid
dutamic acid
Amido thereof
Aldne
Cyitdno
Olydne
I10leudne
budne
Mabionino
Phonyldanine
Prolino
Serine
Thmonine
Tyroiine
11
80
35
41
10
31
29
49
47
49
-
51
88
14
61
31
39
33
29
61
833
I 852
190
2 0 0 -21
2 2 w
44
44
16
44
31
74
38
30
59
916
Frequently, however the differences between heteroenzymes are less obvious. Just as the insulins derived from
beef,hog, sheep,horse, and whaledifferamongeachother
only in terms of three amino acids between the cysteine
residues 7 and 11 of the A chain [26],so the hemopeptides of cytochrome C differ from one another only
in a surprisingly small degree, in spite of very different
origin 126,331.
Chromatographic analysis of cytochrome c has shown
that its composition is nearly the same, whether extracted from horse heart, whale heart or yeast, but that
cytochrome c obtained from Desulfovibrio desulfuricans
differs materially from all otber cytochromes 1341. Recently Anfinsen et al. have published comparative
studies on the composition of ribonucleases derived
from various sources. They report only minor differences
between ribonucleases obtained from pig or beef
pancreas, but appreciable differences between the latter
and those obtained from sheep pancreas [35,361.These
differences were discovered by means of the fingerprint technique referred to above (Fig. 2).
Using this technique, Ingram was able to distinguish
between normal human hemoglobin A and the abnormal
forms C and S, which are only slightly different from
the former. Of the 300 amino acid residues found in one
molecule, one glutamic acid residue in hemoglobin A
[33) J. I. Harris, F. Sanger, and M. A . Naughton, Arch. Biochem.
Biophysics 65, 427 (1956).
[34] K. Takahashi, K. Titani, and S. Minakani, Biochemistry
1 0
3aQ3b
1
4
0
2 0 0 02'
lg0
2 2 0
(b)
Fig. 2. Two-dimensional chromatographic separation of the products
obtained when trypsin and chymotrypsin are allowed to act on oxidized
pancreatic ribonucleasc. (a) Enzyme derived from beef; (b) Enzyme
derived from sheep. Paper chromatography from loft to right (origin
indicated by an arrow in the left margin). Paper electrophorcsia from
top (cathode) to bottom (anode).
Anfinsen er al., J. biol. Chomistry 234, 1118 (1959).
more slowly and is more stable with respect to alkali,
than either the more rapidly migrating sheep enzyme or
the most rapidly migrating beef enzyme [39].Our own
early studies of LDH dealt with the same problem. We
were able to observe differences in electrophoretic
mobility between LDH from beef and hog hearts, and
between LDH from rabbit and rat skeletal muscles [IS].
We were able to cleave these enzymes by means of
trypsin and to compare the resulting peptides by means
of high-voltage electrophoresis [MI. Small, but clearcut differences were observed. Amino acid analyses,
carried out by Dr. W e b e r in the laboratory of Professor
1351
[37] V. M . Ingram, Nature (London) 180,326 (1957).
[38] V. M . Ingram, Lecture, Fourth International Congress of
Biochemistry, Vienna 1958.
1391 A . J. Vithayathil, F. Buck, M. Bier, and F. F. Nord, Arch.
Biochem. Biophysics 92, 532 (1961).
[40] Th. Wieland, G. Ppeiderer, and K . Rajewsky, 2. Naturforsch. ISb, 434 (1960).
172
Angew. Chem. internat. Edit. I Vol. I (1962) I No. 4
(Tokyo)46, 1323 (1959).
C.B. Anfinsen, S. E. G. Aqvist, J. P. Cooke, and B. Jbnsson,
J. biol. Chemistry 234, 11 18 (1959).
[36] A. M.Katz, J. Dreyer, and C. B. Anfinsen, J. biol. Chemistry
234, 2897 (1959).
Brenner in Basle [41], showed that the differences could
be attributed to differences in the primary structure.
Table 3 lists the differences between LDH obtained from
beef and pig heart and from rabbit and rat muscle.
Table 3. Composition of LDH of different origin (Amino acid molecules
per 130000 g.protein)
LDH from
Heart muscle
Beef
Hog
(2 det.)
(2 compon.) 1161
2
1
Lysine
Arginine
Histidine
Skeletal muscle
A
C
97-98
34
27 -28
98
36
28
a7
28
25
86
32
27
Aspartic acid
Glutamic acid
125
115
125
116
126- 127
120
Alanine
Glycine
Isoleucine
Leucine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tyrosine
Valine
70
87
76
128
30
19
45
86
55
25
139
70
87
80
125
33
21
47
92
57
27
132
74
91
79
135
34-35
19-20
43
90
53
27
131
Rat
(2 extreme
values of
4 det.)
Rabbit
-
99
38
21
103
38
23
98
35
37
125
117-118
117
104
119
106
108
106
74
93
80
134
31-32
21
45
88
52
27
132
71
90
83
130
25
25
73
91
a7
134
26
26
47
94
43
26
132
76
94
79
130
32
26
38
74
41
26
126
44
88
42
25
130
At first sight, the distribution of all residues seems similar in
all four products. This distribution is perhaps characteristic
for the “LDH” property of these enzymes. The content of
some amino acids, like alanine, isoleucine, tyrosine and
cysteine, which were determined in up to 14 residues by a
different method in most, but not all, instances, was surprisingly constant. Other residues, notably lysine, aspartic
acid, glutamic acid and threonine, occur in the various enzymes in clearly different amounts. Generally speaking the
enzymes obtained from the heart muscle of different animals
are more like each other than those from skeletal muscle
which in turn are similar. The greater mobility of pig heart
LDH at pH 8.6 with respect to the anode can be attributed
to a lower content of cationic, and a higher content of anionic,
residues. Lactate dehydrogenases obtained from skeletal
muscle contain fewer anionic side chains and therefore tend
to remain behind when subjected to electrophoresis.
Our analytical comparison also included crystalline
LDH obtained from different organs of the same animal.
LDH derived from rat liver and rat muscle were indistinguishable by electrophoresis, but differed from
one another significantly, although only moderately,
in their leucine and valine content. The muscle preparation contained, on the average, 3-5 more moles per
mole of enzyme than the liver preparation. Differences
due to organs of origin are much more clear-cut in the
case of LDH derived from rat skeletal muscle and rat
heart (Band I). As shown in Table 4, these differences
involve all amino acids. In this case as well, the divergence in electrophoretic behavior can be explained
by different charges. Heart LDH No. I, which migrates
rapidly to the anode, contains much more aspartic acid
and less lysine and arginine than the other, electrophoretically less mobile, LDH’s derived from liver and
skeletal muscle. The pronounced organ specificities
[41] Th. Wieland and G. Pfleiderer, Ann. N.Y. Acad. Sci. 94,
691 (1961). Several of the amino acid analyses reported herein
were slightly but not significantly different when repeated with
more highly purified forms of LDH.
Angew. Chem. internat. Edit. / VoI. I (1962)1 No.4
Table 4. Composition of LDH derived from liver, skeletal muscle, and
heart muscle of the rat. (Amino acid molecules per 130000 g. protein).
I
I
Rat LDH from
Eztremesot
103
38
23
95
34-35
26
117
110
117
119
106
141
ioa--109
79
95
82
129
26
30
51
91
47
27
124
71
90
83
100
Aspartic acid
Glutamic acid
Alanine
Glycine
Isoleucine
Leucine
Methionine
Phenylalanine
Proline
Seriae
Threonine
Tyrosine
Valine
Hatt
(Band I.
cf. p. 174)
99
38
21
Lysine
Arginine
Histidine
40
25
124
25
28
48
86
44
26
116
Skeletal muscle
(2 extreme values
of 4 dete
104
130
25
25
44
88
42
25
130
73
91
87
134
26
26
47
94
43
26
132
a1
89
a3
130
33
21
40
92
53
21
134
observed here were also confirmed by immunological
studies. The antibody produced by the chicken against
the rabbit skeletal enzyme, inactivated the rabbit heart
enzyme much less than its antigen (i.e. the muscle
enzyme) [42].
Finally, some reference should be made to the two
malate dehydrogenases which occur in the same cell in
beef heart and rat liver, but originate in different regions
of the cell. A distinction can be drawn between mitochondrial and cytoplasmic malate dehydrogenase[43-451.
This can best be done by electrophoresis on a suitable
membrane where, at pH 8.6, the cytoplasmic dehydrogenase migrates to the anode more rapidly than the
mitochondria1 [46]. The difference seems to be due to the
fact that the latter also has a lipoid component. On
treatment with n-butanol, one enzyme can be transformed into the other [47]. The question now arises
whether in this, so far unique, case the term “heteroenzyme forms” should be applied because the precise
intracellular localization is known, or whether they
should be designated as “multiple forms,” since both
enzymes occur in the same cell type.
Differences Between Multiple Forms of an Enzyme
(Isozymes)
The most important discoveries with regard to multiple
enzyme forms have been described in the preceding
sections from a historical point of view. More recently
similar observations have been made on many other
enzymes, but these cannot be discussed here. The following observations are restricted to examples where multi[42] J. S. Nisselbaum and 0. Bodansky, J. biol. Chemistry 234,
3276 (1959).
[43] G. S. Christie and J. D. Judah, Proc. Roy. SOC. (London) 141,
420 (1953).
[44] A . Delbriick, H. Schimassek, K. Bartsch, and Th. Biicher,
Biochem. Z . 331, 297 (1959).
[45] C. J. R. Thorne, Biochim. biophysica Acta 42, 175 (1960).
[46] Th. Wieland, G. Pjleiderer. I. Haupt, and W. Worner, Biochem. Z . 332, 1 (1959).
[47] A . J. Sophianopoulosand C . S. Vestling, Biochim. biophysica
Acta 45, 400 (1960).
173
ple forms have been clearly separated, or where they
may have been located by visual tests or even studied in
isolated form. In most cases electrophoresis has been
the method of choice, particularly zone electrophoresis.
It has generally been customary to designate those
multiple forms of an enzyme which migrate most rapidly
to the anode as I, the next slower ones as 11,111, etc.
A distinction must be made as to whether the aim is
analysis or micropreparation. For the former, paper
electrophoresis is most suited. Of decisive significancein
this connectionwas the development of methods by which
the separated enzymes could be made visible. Thus,
for DPN-dependent enzymes a very sensitive spray test
was developed which is based on the disappearance of
DPNH fluorescence [Zl]. Many other enzyme reactions
can be coupled with reactions of DPN-specific enzymes,
so that the technique has fairly wide application. In the
case of redox reactions, identification by means of
tetrazolium salts can be very useful; on reduction,
strongly colored formazanes are formed [48,49]. Other
enzymes can be detected by means of histochemical
procedures. Hunter and Markert [SO] have used the
latter to detect enzymes on starch gel electropherograms
(“zymograms”). Vieme [Sl] has developed a spectrophotometric method for the detection in situ of enzymes.
This author allowed the substrate and coenzyme of
LDH to react with the separated enzymes on agar and
then followed the reactions optically.
One of the disadvantages of paper electrophoresis is
that the separated bands of the multiple enzymes cannot
be determined quantitatively on the paper nor can they
be isolated from the paper without losses. However,
elution can be quantitative in the case of synthetic
membranes which, in addition, permit an excellent and
rapid separation of the LDH components at high
voltages (Fig. 3). With this technique 0.01 ml. tissue
extract can be separated in about 1 hour by applying
about 40volt/cm. to a filter saturated with Veronal
buffer; the separated LDH enzymes can be detected
by spraying with a combined coenzyme and substrate
solution using a pressure-type sprayer with suitable
metal orifice; the enzymes appear as sharp, dark bands
and can be eluted directly into cuvettes after the filter
sheet has been cut into strips.
The micropreparative electrophoresis technique employs either starch gel (Smithies [52]), a starch slurry
in a block [53] or a column [54]. When a gel technique
is used, it is best to isolate the enzymes by forcing out
the previously frozen segment by means of a hypodermic syringe. When working with a starch slurry, the
enzymes are allowed to migrate into glass powder [SS].
Ion exchange chromatography is also suitable for the
separation of multiple forms of enzymes. As early as
1953, Stein and Moore [56] succeeded in separating a
crystalline ribonucbase preparation into two active
forms by means of Amberlite IRC-50. Cellulose-type
exchangers are more useful in the case of especially
sensitive proteins ; a good example is diethylaminoethylcellulose, DEAE [57]. According to Hess [58] LDH
isozymes derived from different organs can be separated
on such a cellulose column by means of fractional elution
with buffers of varying pH and NaCl content.
On the basis of our collaborative work with H. Determann and F. Ortanderl we have found that better results
are obtained by using DEAE Sephadex exchanger, which
has a much higher capacity. Figure 4 shows the separation of a uniformly crystalline rat heart enzyme into
E
St
fa)
fb)
e-
-a
10
5
m
I
20
30
40
50
55
E
Fig. 4. Chromatographic separation of isozyme from crystalline rat
heart LDH by means of DEAE Sephadex. Elution with 0.05 N phosphate buffer (pH 6) in NaCl up to 0.5 M. Component E is the last to
appear in the eluate.
Ordinate: Deflection of the recorder in the flow photometer (Uvicord)
Abscissa: Fraction number.
four components. Elution was carried out with a phosphate buffer (PH 6) with increasing NaCl concentration.
In accordance with the order of appearance in the eluate,
the separate multiple forms obtained on chromatography are designated as A, B, C, etc. This last procedure
appears the most suitable for the analytical and preparative separation of multiple enzyme forms.
[52]0. Smithies, Biochem. J. 61, 629 (1955).
[53]H. G.Kunkel and R . J. Slater, Proc. SOC.exp. Biol. Med. 80,
42 (1951).
[54]P. Flodin and J. Porath, Biochim. biophysica Acta 13, 175
(1954).
[55]Th. Wieland, G.Ppeiderer, and H. L. Rettig, Angew. Chem.
70,341 (1958).
I561 C.H. W.Hirs, S. Moore, and W,H. Stein, J. biol. Chemistry
200, 493 (1953).
1571 E. A. Peterson and H. A . Sober, J. Amer. chem. SOC.78,
751 (1956).
[58]B. Hess and S. I. Walter, Klin. Wschr. 38, 1080 (1960).
174
Angew. Chem. internat. Edit. ]-VoI.I (1962)I No. 4
Lactate Dehydrogenases (LDH)
Lactate dehydrogenases have been studied in detail.
As early as 1956 we reported five different LDH components isolated from various organs of the rat by
means of paper electrophoresis [21]. Subsequently we
succeeded in establishing heterogeneity in other enzymes
from animals and man [46, 591. These studies soon led
to several questions:
1. Do all organs contain the same number of LDH
isozymes ?
2. Is the distribution of total activity with respect to the
individual components the same in all organs?
3. What are the chemical and physical differences?
4. Is the primary structure of electrophoreticallysimilar
lactate dehydrogenases derived from different organs
the same?
The answer to the first question is related to the state
of development of the analytical technique. Formerly we
considered the pig heart LDH as one of the few lactate
dehydrogenases which occur in the organ as a singlecomponent enzyme. However, when electrophoresis was
carried out on synthetic membranes, a second component was found, even if the preparation was crystalline; this second component amounted to only 1 % of
the total. The situation was similar for the LDH from
rat skeletal muscle. This enzyme was formerly considered
as a pure substance, though quite different from heart
muscle LDH. We now know that both LDH preparations
contain five isozymes, four of which however contain
only a fraction of the total activity present (I-1V).
Nevertheless, it is reasonable to assume that the number
of LDH components is not the same in all organs of an
animal.
Concerning the second question, it may be said that
large differences exist in the enzyme distribution patterns
of various organs. Table 5 lists the quantities (as percentages) of isozymes in the organs of the rat and of
man. As can be seen, there exist undoubtedly two types
of distribution in organs, with most of the activity in man
or mammal occurring in either the fist or the last iso-
zyme. Studies on the age dependence of this distribution
have shown that this polarization of distribution does
not occur in man while the organs are still in the
embryonic stage; rather all multiple forms show a
similar distribution with the main emphasis on component I1 [60].These differences between the embryonic
and the adult stages were also observed in other mammals [23,61].
In organs of adults the patterns of distribution are quite
constant and characteristic. Heart muscle and liver are
extremes. Normally the blood serum contains only a
minimal amount of LDH activity, which originates from
the rapidly migrating LDH forms of the erythrocytes. If,
however, heart muscle or liver has become damaged, the
corresponding isozyme will occur in the blood serum
in considerably increased amounts. Their analysis may
therefore provide a useful diagnostic test [61a]. According to Hess and W a l t e r [62] it is possible to completely adsorb onto DEAE cellulose (anion exchanger)
the heart LDH (I) occuring in blood as a result of a
coronary infarct, whereas the liver LDH, freed as a
result of hepatitis, cannot be adsorbed by stirring with
the anion exchanger [62].
With reference to the third question it may be stated
that differences between the isozymes of LDH were first
observed in the course of electrophoretic studies. They
are therefore the certain result of different net charges
of the protein molecule. Moreover, in proteins migrating
with different speeds, relationships have been established
between electrophoretic behavior and other properties.
Thus, in the case of isolated isozymes obtained from
rat kidney, the effect of sulf~tein inhibiting reactions
increases as the negative charges on the proteins increase
in number [20]. For example, Band I may, under certain
conditions, be inhibited by as much as 80 %, whereas
Band V, under the same conditions, may be inhibited by
no more than 50%. This means that the affinity of
coenzyme and sulfIte for enzyme I is greater than for
enzyme V. It was shown subsequently that Bands I1 to
Table 6. Relationship between electrophoretic mobility and other
physical or chemical properties of the five multiple forms of mammalian
lactate dehydrogenase
LDH
Table 5. Distribution of the average activity of LDH
components in various organs from man and the rat
Organ
I
(Enzyme no.)
I ManI I1
Heart muscle
Kidney
Cerebrum
Cerebellum
Erythrocytes
Liver
Skeletal muscle
Epidermis
60
28
28
39
36
0.2
3
0
Heart muscle
Kidney
Brain
Skeletal muscle
Liver
34
36
25
0.5
0
I
111
30
34
32
34
35
0.8
4
0
5
21
19
20
9
1
7
4
43
27
23
1
0
15
7
18
1.5
0
I IV I v
2
6
5
Heat stability (half-time
of denaturation at 50 "C)
-
-
4
10
17
94
76
79
Optimal pyruvate
concentration [mole/I.] 1.2 10-3
+ 0.1510-3
Increase in reaction rate
with 10 "Cincrease
-+ 2.1
5
9
24
3
3
2
20
10
94
97
3
11
16
7
Rat
[59] Z. Haupt and H. Giersberg, Natunvissenschaften 45, 268
(1958).
Angew. Chem. internat. Edit. I VoI. I (1962)1 No. 4
1.5
1601 G. Pfleiderer and E. D. Wachsmuth, Biochem. Z . 334, 185
(1961).
[61]L. B. Flexner, J. B. Flexner, R. B. Roberts, and ii. De la
Haba, Devel. Biol. 2, 313 (1960).
[61-a] For complete description: F. W. Schmidt et al. in H. U.
Bergmeyer: Methoden der enzymatischen Analyse. Verlag C h e
mie, Weinheim/Bergstr. 1962.
[62] 8. Hess and S . Z. Walter, Klin. Wschr. 39, 213 (1961).
175
IV, which migrate at intermediate speeds, also have
intermediatesensitivityto sulfite [63]. Table 6 reproduces
this relationship. At the same time it was observed that
other properties of the enzyme also undergo change to
the same extent 164,651. Thus, the optimum pyruvate
concentration for I as compared to V drops by a factor
~ M to 1 . 5 10-4
~ M); the
of ten (i.e. from 1 . 2 10-3
temperature coefficient increases uniformly from 1.5
for V to 2.1 for I. The differences are particularly large
as far as the heat stability of various proteins is concerned: The half-time value for denaturation at 55 "C
amounts to practically infinity for I, 100mins. for 11,
40 mins. for 111, 10 mins. for IV, and 3 mins. for V.
Perhaps the relationship between such diverde physical
data may ultimately provide a deeper insight into the
mechanisms of adsorption and reaction with substrate
and coenzyme.
The differences in charge between isozymes may be
due to the fact that the more rapidly migrating protein
contains fewer side chains, perhaps because of acylation
of a part of the &-aminogroups of lysine; or they may
contain more carboxylate side-chains than the more
slowly migrating proteins, where some carboxyl groups
have been neutralized by amide formation. Of the
several examples of this type attention is called to two
mushroom poisons, a- and p-amanitine [66]. The
difference in charge may also be due to chemically
bound phosphate, sulfate or other polycharged residues.
According to our analyses, differences in phosphate
content can be ruled out. Differences in mobility in the
electrical field may also be due to differences in heavy
metal content. On the other hand, the differences in
structures may be much more profound and may go as
far as differences in amino acid composition, i.e. differences in the primary structure. A clear-cut answer can
be given only on the basis of the chemical analysis of
components, a procedure so far carried out for only a
few compounds. A comparison was made with the LDH
isozymes 1-111 obtained from rat heart by means of
preparative starch electrophoresis, or chromatography
on DEAE Sephadex. Differences in composition then
became more evident. The pronounced drop in aspartic
acid content from I to I11 might explain why these compounds differ in their rate of migration in an electric
field (Table 7). On the other hand, no difference is apparent between LDH I and I1 from beef heart (cf. values in Table 3). As for the molecular weights, no
differences were observed in the preparations so far
studied. Moreover, the observation that many isozyme
mixtures precipitate uniformly in the ultracentrifuge
would also seem to suggest a uniform molecular weight.
The situation is similar as far as the fourth question is
concerned. The only fact that is certain is that the LDH
isozymes V from all rat organs behave in the same
[63] Th. Wieland, G. Pfieiderer, and F. Ortanderl, Biochem. Z .
331, 103 (1959).
1641 P. W. G. Plagemann, K . F. Gregory, and F. Wroblewski, J.
biol. Chemistry 235, 2288 (1960).
[65] P. W. G. Plagemann, K . F. Gregory, and F. Wroblewski,
Biochem. 2. 334, 37 (1961).
1661 Th. Wieland and W . Boehringer, Liebigs Ann. Chem. 635,
178 (1960).
176
fashion upon electrophoresisand that they are inhibited
by sulfite to the same extent. Notwithstanding this, the
LDH isozymes V obtained from skeletal muscle and
Histidine
26
Aspartic acid
Glutamicacid
141
108-109
Alanine
81
Glycine
89
Isoleucine
83
Leucine
130
Methionine
33
Phenylalanine 21
Proline
40
Serine
92
Threonine
52-53
27
Tyrosine
Valine
134
25
25-26
136
109
130
111
79
89
85
131
32
22
45
87
50
26
134
81
93
83
129
29
26
46
89
49
27
131
liver are not identical (cf. Table4). Comparisons of
other isozymes from different organs have not yet
been made but are being planned, now that convenient
and effective methods for chromatographic separation
with DEAE Sephadex are at hand.
MuItipIe Forms of Other Enzymes
In recent years the number of times that multiple forms
of other enzymes have been reported has risen considerably. In some instances several bands were observed
following electrophoretic separation; however, these
were neither isolated nor characterized further. We shall
now consider some examples where detailed studies
have brought to light new points of view.
Among animal enzyme studies mention should be made
of a thorough investigationdealingwith the heterogeneity
of tissue dehydrogenases in the rat. Following zone
electrophoresisin starch gel, the strips were incubated in
solutions that contained the specific substrate, a tetrazolium salt, methylene blue and the specific pyridine
nucleotide. Direct staining led to the identification of
the isozymes of lactate, malate, isocitrate, glucose-6phosphate and a-glycerophosphate dehydrogenases,
each of which gave rise to a different number of bands
depending on the organ of origin [67].
A recent observation made on pancreatic ribonuclease,
the heterogeneity of which has already been referred to,
indicates a closer relationship of these isozymes: Dickman, Morell, and Triipin [68], working with mice and
beef pancreas, chromatographed their material on
Amberlite IRC-50 and obtained three components when
the glands were extracted either with sucrose or phosphate solutions. The two original isozymes were ob[67] M .
U.Tsao, Arch. Biochem. Biophysics 90,234 (1960).
[68] S. R. Dickman, G. A . MorriIl, and K . M . Trupin, J. biol.
Chemistry 235, 169 (1960).
Angew. Chem. internat. Edit. I Vol. I (1962) I No. 4
served when extraction was carried out with 0.25 M
sulfuric acid. It turned out that the additional naturally
occurring components, which can be obtained by chromatography, were transformed into the other two components by treatment with sulfuric acid; at the same
time total activity was enhanced; this suggested the
properties of a zymogen or of a ribonucleasemodified by
an inhibitor.
According to Meister et al. [69] the crystalline 1-amino
acid oxidase isolated from snake venom (Crotalus
adamanteus) consists of two components of equal
sedimentation velocity and equal specific activity which
can be separated electrophoretically. When pooled
venom was worked up, an additional component of
equal,effectiveness was observed. This indicates quite
clearly that comparisons are best carried out with
material obtained from the same starting material. The
ideal procedure would be to study the products of
geneticallyuniform cells propagated in vitro, but present
methods of culture do not yet allow this.
Clearer results are obtained from studies with unicellular
organimss, as shown below. As early as 1937 Malmstrom [70], using zone electrophoresis and ion exchange
chromatography, isolated four proteins with enolase activity from yeast. Similar studies were carried out in our
laboratory in recent years. Thus, Wiirner [71] was able to
demonstrate the existence of, and quantitatively determine, five enolase isozymes isolated from brewer’s yeast
by means of electrophoresis on synthetic membranes.
Uniform strains isolated from single-cellculture yielded
the same number of enolase components, but with different activity distributions. This contrasts with baker’s
yeast which contains {onlya single enolase-like protein
component.
Similarly the multiplicity of hexokinases from yeast,
described in the very impressive, previously cited studies
of Trayser and Colowick [28], is preserved in uniform
single-cell cultures.
In recent years the occurrence of multiple forms of
different enzymes has been reported in bacteria, particularly for E. coli. Thus, at least two threonine deaminases
(TD) were demonstrated in E. coli extracts in 1957.
The first, the so-called biosynthetic TD which utilizes
a-ketobutyric acid, is needed for the biosynthesis of
isoleucine. It can be inhibited by I-isoleucine and contains pyridoxal phosphate as coenzyme [72]. The second
TD, already described by Wood and Gunsalus [73],
also requires pyridoxal phosphate as well as adenosine
triphosphate and glutathione, in order to develop
maximum activity. It cannot be inhibited by isoleucine.
The question arises here whether these enzymes can in
fact be considered multiple forms, since their coenzyme
requirement differs.
There can be no doubt, however, of the fact that tha
recently discovered aspartokinases of E. coli [Stadman
[69]D. Wellner and A . Meister, J. biol. Chemistry 235,2013(1960).
[70]B. G. Malmstriim, Arch. Biochem. Biophysics 70, 58 (1957).
[71]A. Wiirner, unpublished results.
[72] H. E. Umbarger and B. Brown, J. Bacteriol. 73, 105 (1957).
[73] W. A. Wood and I. C. Gunsalus, J. biol. Chemistry 181,171
(1949).
Angew. Chem. internat. Edit. 1 Vol. I (1962) No. 4
et al. [74] can be considered as being isozymes, two of
which may be isolated by ammonium sulfate precipitation. The phosphorylation of a P-carboxyl group of
aspartic acid by ATP and a kinase (aspartokinase) is a
fundamental reaction in the biosynthesis of lysine,
threonine and methionine in E. coli. As shown by the
scheme below, this synthesis proceeds by way of two
common steps which then diverge. One of these two
aspartokinases was inhibited specifically and noncompetitively by L-lysine. When E. coli was grown in
10-2 M lysine solution, the formation of this enzyme
was completely inhibited (repression).
HOzC-CH-CHz-COzH
I
NHz
aspartic acid
ATP
-1 +(aspartokinase)
HOzC-CH-CHz-COOPO~HZ
I
NHz
P-aspartylphosphate
-1 TPNH
HOZC-CH-CHz-CHO
--f
I
+ --f lysine
NHz
P-semialdehyde
4
TPNH
HOzC-CH-CH2-CH~OH
--f
I
+-+
threonine
NHz
homoserine
1
HOZC-CH-CHZ-CH~-SCH,
I
NHz
methionine
The second aspartokinase is inhibited specifically by Lthreonine. The biological significance of these isozymes
may be that overproduction is prevented by the specific
inhibition due to agiven end-product (feed-backconuol).
So far there is no proof for the existence of a similar
feed-back function for any of the other isozymes discussed here.
Concluding Remarks
It is necessary to regard the now well-proved chemical
differences between many enzymes of similar action as
an expression of the development of the organism. Just
as organisms are likely to be differentiated into their
multiple forms in the course of history, perhaps starting
with one cell, so the composition of their proteins may
also have undergone differentiation. This fact, in so far
as it implies visible and concrete differences, has been
known for sometime; an example is mammalian aglobulin, which gives rise to differentantibodies in different species, a fact that becomes evident when immunization is carried out improperly. It is also well
known that the hemoglobins of the many species studied
to-date exhibit distinct differences [75]. The hope that
in this domain the fingerprint technique would become
[74]E. R . Stadtman, G. N. Cohen, G. Le Bras, and H. de Robichon-Szulmajster, J. biol. Chemistry 236,2033 (1961).
[75]H. Neurath and K. Bailey: The Proteins. Academic Press,
New York 1954,Vol. 11, p. 317.
177
a tool of sharp differentiation has been dampened somewhat by the results of comparative studies camed out
by Pauiing et al. [76]. These investigators were unable
to find any difference between the hemoglobins of man,
gorilla, and chimpanzee by this method.
Hemoglobin also exhibits heterogeneity, i.e. it can be
separated into multiple forms by means of zone electrophoresis [77] or chromatography on Amberlite IRC-50
[78,79]. This has been studied particularly intensively
in the ferric cyanohemoglobin of man [79]. Two components, A1 and A,,, were regularly produced in an
approximate ratio of 1:9. They differ from one another
in that A,,, does not contain isoleucine. Fetal hemoglobin F can also be separated into two components.
The amino acid sequence in myoglobin obtained from
the sperm whale has been studied both analytically [SO]
and by X-ray crystallography “1. Five components
[76] E. Zuckerkandt, R . T. Jones, and L. Pauling, Proc. nat. Acad.
Sci. USA 46, 1349 (1960).
[77] H. G. Kunkel and G. WaIIenius, Science (Washington) I22,
288 (1955).
[78] M. Morrison and J. L. Cook, Science (Washington) 122,920
(1955).
[79] D. W. Allen, W. A. Schroeder, and J. BaIog, J. Amer. chem.
SOC.80, 1628 (1958).
[80] A . B. Edmundson and C., H. W. Hirs, Nature (London)
190, 663 (1961).
[81] J. C. Kendrew, H. C. Watson, B. E. Strandberg, E. R. Dickerson, D. C. Phillips, and V. C. Shore, Nature (London) I90,
666 (1961).
were isolated by chromatography on Amberlite IRC-50.
It is believed that these have the same amino acid composition. However, chemical comparisons of proteins
should not be restricted merely to a determination of
the total number of amino acids present. Akeson and
Theorell [82] have examined two myoglobins isolated
from horse muscle and their work shows that only a
competent finger-print analysis, or a similarly effective
procedure, will ensure that differences in primary
structure will be detected in proteins of similar composition.
Thus it can be seen that the occurrence of multiple
forms is not restricted to the enzymes discussed in this
review. Quite possibly many more natural proteins than
have so far been studied exist in isofunctional units.
Such proteins can be designated as “homoproteins.”
Perhaps the concept advanced by geneticists that the
formation of each type of protein requires its specific
gene may have to be modified if it turns out that differences in composition extend to appreciable deviations
in the amino acid sequence. Undoubtedly future biochemical research will have as one of its important aims
the clarification of such structural differences and their
possible biological significance.
Received, November 2nd. 1961
[A 173/16 IE]
[82] A. Akeson and H.Theorell, Arch. Biochem. Biophysics 91,
319 (1960).
Conversion of Natural Substances by Microbial Enzymes [*I
BY PROF. DR. CH. T A W
INSTITUT FUR ORGANISCHE CHEMIE DER UNIVERSITAT BASEL, SWITZERLAND
Micro-organisms form enzymes that catalyze many reactions which are diffic.lt to carry
out in the laboratory or which involve many steps. Enzymes even react with substances
which are normally foreign to them. Thefrequently high stereospecificity of microbiological
reactions is of particular significance. The most important types of reactions are: hydroxylations, dehydrogenations (oxidations), and hydrogenations (reductions), cleavage of ester
and C-C bonds, and transglycosidations. The variety of the compounds converted is extremely great.
1. Introduction
2. Methods
3. Steroids
(a) Hormones of the Androstane and Pregnane Series
(b) Estrogens
(c) Bile acids
(d) Cardenolides and Bufadienolides
4. Terpenes
5. Phenols
1. Introduction
Micro-organisms seem to compensate for their lack
of variety of external forms by specific internal chemism,
a criterion which sets them apart fram higher plants,
which have a differentiatedmorphology. Their metabolic
178
6.
7.
8.
9.
Carbohydrates
Antibiotics
Alkaloids
Nature of the Enzymes. Reaction Mechanisms
(a) Hydroxysteroid Dehydrogenases
(b) A-Dehydrogenases and A-Reductases
(c) As-3-Ketosteroid Isomerase
(d) Hydroxylases
10. Conclusion
products (metabolites) cover the entire spectrum of
chemical structures; these may be very simple, or very
complex, compounds. This is illustrated in a most
impressive manner by the numerous antibiotics discovered during the last decades. Studies of mould
[*I Extended version of lectures delivered in Braunschweig (1960),
Berlin (1960), and Base1 (1961).
Angew. Chem. internat. Edit. 1 VoI. 1 (1962)1 NO.4
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