80
3 The Structural Variety and Metabolism of Proteins
this has best been shown for enolases 2 and 3 of
the roundworm Ascaris suum [81]. The apparent
presence of multiple protein forms can be due to
different artefacts. Particularly in the case of
post-translationally modified proteins, forms with
altered electrophoretic mobilities can occur during extraction as the result of both enzymatic and
non-enzymatic processes. Because of overlapping
substrate specificities, assumed enzyme-specific
staining of the electrophoregram can detect other
proteins, for example in the case of alcohol
dehydrogenase and lactate dehydrogenase in
Drosophila melanogaster; each of these can react
with the substrate of the other.
In most cases, there are functional differences
between multiple enzyme forms, e.g. in specific
activity, substrate specificity and affinity, pH optimum, regulatory characteristics, temperature
dependence and stability. These differences are
generally much greater between the products of
different genes, and between some posttranslationally modified enzymes, than between
alleloenzymes. The immunological similarities
between the latter are also generally maintained,
so that, for example, enzymatically inactive null
variants can be detected immunologically. The
existence of isoenzymes was considered from the
outset to represent a fine adaptation to different
functional conditions. The idea of an adaptive
role is supported by the variable distribution of
isoenzymes between different tissues and cell
compartments, as well as by the fact that they
appear in a strict order during ontogeny. On the
other hand, it should be noted that gene duplication and subsequent diversification, according to
our present knowledge, are important mechanisms in the evolution of enzymes; thus, some
isoenzymes may only be intermediate steps in the
evolutionary transformation of proteins and may
themselves have no adaptive value.
There is an abundance of plausible examples of
the adaptive significance of isoenzymes, although
in many cases these are not without a rider. For
example, the activity of aldolase A of mammalian
muscle is five times higher with fructose-1,6bisphosphate than with fructose-1-phosphate,
whereas both substrates are used with equal efficiency by aldolase B in the liver; in this way, the
muscle enzyme is adapted to glycogen degradation, and the liver enzyme to the utilization of
fructose and glycogen synthesis. However, this
does not explain why, in the brain, there is an
aldolase C with intermediate specificity. The typicallactate dehydrogenase (LDH) isoenzyme of
mammalian muscle ~ is much less inhibited by
pyruvate than is the typical heart isoenzyme B4; if
one assumes that isoenzyme ~ is adapted to
anaerobic lactate production in muscle, and
isoenzyme B4 to aerobic lactate oxidation in the
heart, it is then difficult to understand why the
usually aerobically active liver cells in many mammalian species contain so much a4 [182]. The
existence of different isoenzymes, e.g. of malate
dehydrogenase, aspartate transaminase, malate
enzyme and NADH-specific isocitrate dehydrogenase, in mitochondria and the cytoplasm is
plausible in view of the different concentrations
of metabolites and the opposite directions of the
reactions in these different compartments. But
one should also consider the fact that the mitochondrial enzymes are made on cytoplasmic ribosomes and must then be transported into the
mitochondria; this requires special protein structures. It is also clear that the enzymes of the lysosomes and membrane-bound enzymes must be
different from those of the cytoplasm; this has
been shown, for example, for various glycosidases and phosphatases [182].
3.2 Protein Synthesis
Since the first successful attempts at the beginning of the 1950s, cell-free protein synthesis systems have been established from several very different eukaryotic cells. Significantly higher yields
of transcription and translation products are to be
obtained, however, following injection of DNA or
mRNA into whole, living cells. The developing
oocytes of the clawed frog, Xenopus laevis, are
very often used for this purpose; because of their
large size (a diameter of 1.2 mm) they can yield
up to 100 times more product than other cells.
Such in vitro or in vivo systems can also correctly
translate foreign genetic material; the cytoplasmic components of protein biosynthesis are
apparently very similar in all eukaryote cells.
Extremely conservative evolution of the proteinsynthesizing machinery is to be expected because
each large change would have catastrophic consequences for the cell. Only in the prokaryotes and
in eukaryote mitochondria, which are assumed to
have arisen from endosymbiotic prokaryotes, are
significantly different translation mechanisms to
be found. In spite of the universality of protein
synthesis, the translation of heterologous mRNA
is often markedly reduced in contrast to that of
homologous mRNA; the variable inhibitor sensitivity of the translation of different mRNAs also
3 The Structural Variety and Metabolism of Proteins
this has best been shown for enolases 2 and 3 of
the roundworm Ascaris suum [81]. The apparent
presence of multiple protein forms can be due to
different artefacts. Particularly in the case of
post-translationally modified proteins, forms with
altered electrophoretic mobilities can occur during extraction as the result of both enzymatic and
non-enzymatic processes. Because of overlapping
substrate specificities, assumed enzyme-specific
staining of the electrophoregram can detect other
proteins, for example in the case of alcohol
dehydrogenase and lactate dehydrogenase in
Drosophila melanogaster; each of these can react
with the substrate of the other.
In most cases, there are functional differences
between multiple enzyme forms, e.g. in specific
activity, substrate specificity and affinity, pH optimum, regulatory characteristics, temperature
dependence and stability. These differences are
generally much greater between the products of
different genes, and between some posttranslationally modified enzymes, than between
alleloenzymes. The immunological similarities
between the latter are also generally maintained,
so that, for example, enzymatically inactive null
variants can be detected immunologically. The
existence of isoenzymes was considered from the
outset to represent a fine adaptation to different
functional conditions. The idea of an adaptive
role is supported by the variable distribution of
isoenzymes between different tissues and cell
compartments, as well as by the fact that they
appear in a strict order during ontogeny. On the
other hand, it should be noted that gene duplication and subsequent diversification, according to
our present knowledge, are important mechanisms in the evolution of enzymes; thus, some
isoenzymes may only be intermediate steps in the
evolutionary transformation of proteins and may
themselves have no adaptive value.
There is an abundance of plausible examples of
the adaptive significance of isoenzymes, although
in many cases these are not without a rider. For
example, the activity of aldolase A of mammalian
muscle is five times higher with fructose-1,6bisphosphate than with fructose-1-phosphate,
whereas both substrates are used with equal efficiency by aldolase B in the liver; in this way, the
muscle enzyme is adapted to glycogen degradation, and the liver enzyme to the utilization of
fructose and glycogen synthesis. However, this
does not explain why, in the brain, there is an
aldolase C with intermediate specificity. The typicallactate dehydrogenase (LDH) isoenzyme of
mammalian muscle ~ is much less inhibited by
pyruvate than is the typical heart isoenzyme B4; if
one assumes that isoenzyme ~ is adapted to
anaerobic lactate production in muscle, and
isoenzyme B4 to aerobic lactate oxidation in the
heart, it is then difficult to understand why the
usually aerobically active liver cells in many mammalian species contain so much a4 [182]. The
existence of different isoenzymes, e.g. of malate
dehydrogenase, aspartate transaminase, malate
enzyme and NADH-specific isocitrate dehydrogenase, in mitochondria and the cytoplasm is
plausible in view of the different concentrations
of metabolites and the opposite directions of the
reactions in these different compartments. But
one should also consider the fact that the mitochondrial enzymes are made on cytoplasmic ribosomes and must then be transported into the
mitochondria; this requires special protein structures. It is also clear that the enzymes of the lysosomes and membrane-bound enzymes must be
different from those of the cytoplasm; this has
been shown, for example, for various glycosidases and phosphatases [182].
3.2 Protein Synthesis
Since the first successful attempts at the beginning of the 1950s, cell-free protein synthesis systems have been established from several very different eukaryotic cells. Significantly higher yields
of transcription and translation products are to be
obtained, however, following injection of DNA or
mRNA into whole, living cells. The developing
oocytes of the clawed frog, Xenopus laevis, are
very often used for this purpose; because of their
large size (a diameter of 1.2 mm) they can yield
up to 100 times more product than other cells.
Such in vitro or in vivo systems can also correctly
translate foreign genetic material; the cytoplasmic components of protein biosynthesis are
apparently very similar in all eukaryote cells.
Extremely conservative evolution of the proteinsynthesizing machinery is to be expected because
each large change would have catastrophic consequences for the cell. Only in the prokaryotes and
in eukaryote mitochondria, which are assumed to
have arisen from endosymbiotic prokaryotes, are
significantly different translation mechanisms to
be found. In spite of the universality of protein
synthesis, the translation of heterologous mRNA
is often markedly reduced in contrast to that of
homologous mRNA; the variable inhibitor sensitivity of the translation of different mRNAs also