From Dogfish to Dogs:
TrimethylaminesProtect
Proteins from Urea
George N. Somero
Filling the “osmotic
gap”
and offsetting
the threat
of urea: problems
for cells
of the renal papilla
of approximately
630 mosmol/kg
in
papilla cells of antidiuretic
rats (2).
The osmotic gap problem facing
cells of the renal papilla is distinct
from a second critical problem that
arises from the nature of the solutes
in the extracellular
fluids. The solute that makes the major contribution to the osmolarity
of the extracellular fluids is urea, a strong perturbant
of protein
structure
and
function. Urea concentrations
in the
urine of mammals
not under water
stress are often near 0.3-0.5 M, and
under extremes of water stress, as
might occur in desert rodents, urea
concentrations
may rise to above 4
M (4). Urea diffuses so readily across
cellular
membranes
that the urea
concentrations
in the cells of the
renal papilla are almost certainly
close to, if not identical
with, the
urea concentrations
of the extracellular fluids. And, even at the concentrations of urea found in the absence of water stress, urea is able to
affect significantly
the abilities
of
proteins to function
normally
(7).
Unless the proteins within the cells
of the renal papilla are in some way
“urea adapted,” some means must be
available to protect them from the
perturbing influences of urea.
How, then, have mammals solved
these two problems, the filling of the
osmotic gap and the elimination
of
urea perturbation
of proteins? The
The high osmolarity
and the solute
composition of the extracellular
(tubular and interstitial)
fluids of the
mammalian
kidney present two serious problems for cells of the renal
papilla. First, because of the high
osmotic concentration
of the extracellular fluids, the cells of the renal
papilla must maintain
a high intracellular osmolarity
(at least by vertebrate standards) if they are to
avoid loss of water and consequent
shrinkage.
The
low-molecular-weight
osmotic agents (osmolytes)
used to
build up intracellular
osmolarity
in
the renal papilla have remained
a
subject of controversy (2). It is clear
from recent work that the inorganic
ions, sodium, potassium, and chloride, are not highly concentrated
in
these cells. It seems likely, therefore,
that low-molecular-weight
organic
osmolytes may fill the “osmotic gap”
represented by the difference between total intracellular
inorganic
ion concentrations
and the osmolarity of the extracellular
fluids, a gap
George
N. Somero
is the Iohn Dove lsaacs
Professor of Natural
Philosophy,
Scripps Institution of Oceanography,
University
of Calijornia at San Diego, La lolla, CA 92093.
0886- 17 14/86 S 1.30 0 1986 Int. Union
Physiol.
Scl/Am.
Physlol.
Sot.
Methylamine
counteraction
of urea’s effects
A clue to an adaptive strategy that
might be exploited by mammals to
solve these two problems
comes
from studies of the urea-rich marine
cartilaginous
fishes (sharks, rays,
skates, and holocephalans)
and the
“living fossil,” coelacanth (Latimeria
chalumnae). These fishes have body
fluids in osmotic equilibrium
with
seawater, and they contain urea at
concentrations
of 0.3-0.5 M, i.e., at
levels similar to those found in the
fluids of the mammalian
kidney.
Two ways of coping with the perturbing effects of urea on the *proteins of these urea-rich fishes have
been discovered (7). Some proteins
of these fishes are urea adapted, in
the sense that these proteins display
appropriate
values for kinetic and
structural
properties
only when
physiological
concentrations
of urea
are present. However, for most of the
proteins
so examined,
a second
adaptive strategy has been found.
We have termed this strategy the
“counteracting
solute” strategy to
denote that the protection
of proteins from perturbation
by urea derives from the occurrence within the
cells of these fishes of organic solutes
that can counteract
the effects of
urea on proteins.
The intracellular
fluids of urearich fishes have been known for
many years to contain a set of methylamine solutes, principally
trimethylamine-N-oxide
(TMAO) and glytine betaine (Fig. l), at a total concentration of approximately
one-half
the concentration
of urea (7). Until
recently, however, the raison d’etre
for the use of these particular solutes
and the significance of the 2:1 ratio
of [urea] to summed [methylamines]
were not known.
Our studies of the influences of
these methylamine
compounds
on
proteins appear to explain the adaptive significance of their accumulation and the importance
of the 21
ratio of [urea]: [methylamines].
For
Volume
1/February
1986
NIPS
9
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The accumulation
of methylamine
solutes to counteract
perturbation
of
proteins by urea seems to be an important
and phylogenetically
widespread phenomenon.
In the renal papilla
of the mammalian
kidney, the accumulation
of methylamines
appears tosolve two distinct
problems. The “osmotic gap” may be largely filled by methylamines
and urea, and, because methylamines
appear to be accumulated
to
about one-half the concentration
of urea, they are likely to offset fully
the influences of urea on intracellular
proteins. How the kidney cells
accumulate
and retain methylamines
and how they adjust methylamine
concentrations
in the face of fluctuations
in urea levels remain
fascinating
and important
questions for physiologists
to resolve.
answer appears to be that both problems have been solved by a single
mechanism,
one that is evolutionarily ancient and, possibly, phylogenetically widespread.
CH 3
CH3-I;+=
OI
CH 3
CH
0l-b3
I
CH3-N'CH2-CH2-0-P-0-CH2-C-CH20H
II
I
0
CH3
CH3
‘+
CH3-N-CHCOOI
CH3
Trimethylamine-N-Oxide
Glycine
Glycerylphosphorylcholine
Betaine
(TmO)
FIGURE
1.
Structures of 3 trimethylamine
(GPC)
solutes that play important
NIPS
osmotic roles.
bation of protein function, these data
show that the counteracting
influences of urea and the methylamines
are not manifested only by proteins
from urea-rich fishes. Instead, the
effects of these nitrogenous
solutes
are independent
of the species
source of the protein. These effects
do not, therefore, owe their existence to special adaptations
of the
proteins of urea-rich fishes to urea
and methylamines
but instead are
due to ubiquitous aspects of proteinwater-solute
interactions
discussed
below.
It is appropriate to emphasize that
the accumulation
of only methylamines would not be an advantageous
strategy. Glycine
betaine, TMAO,
and other methylamine
compounds
could, in the absence of urea to
Volume 1/February 1986
-8
-4
0
counteract
their effects, shift the
functional and structural properties
of proteins too far in the direction
opposite from urea-perturbed
proteins For example, the highly stabilizing effects of methylamines
on
protein structure might make proteins too rigid to permit the critical
changes in protein
conformation
that are known to accompany catalysis and regulation. Suffice it to say
that maintaining
the 21 ratio of
[urea]:[methylamines]
is an essential component of the counteracting
solute strategy.
From dogfish to dogs:
methylamines in the
mammalian kidney
The apparent importance
of the
counteracting
solute strategy for
PIG KIDNEYARGININOSUCCIN
ATE
LYASE
84-THORNBACK
RAY LIVERA
ARGININOSUCCINAT
LYASE
J
A
400mM urea ~0.0663
Urea+TMAO
00.0843
Control
0 0.0949
200mM TMAOo0.1046
400mMurea
UreatTMAO
4
8
16
24
~0.0381
00.0439
200mMTMAOo0.0492
-8
-4
0
4
8
16
(md)
[ArgSuc)-' (mM")
[ArgSu$
RECIPROCAL OF SUBSTRATECONCENTRATION
2. Influences of urea, trimethylamine-N-oxide
(TMAO), and a 2:l ratio of [urea]:
(TMAO) on activity (maximal velocity, V,,,) of the urea cycle enzyme, argininosuccinate
lyase, from pig kidney and from thornback ray liver (8).
FIGURE
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.2 on June 16, 2017
a wide spectrum of functional
and
structural properties of proteins, including substrate and cofactor binding abilities, rates of enzymatic activity, and protein conformational
stability,
the methylamines
were
shown to counteract the perturbing
effects of urea, provided that a 21
ratio of [urea] to summed [methylamines] was employed.
That is, the
opposing effects of urea and the
methylamines
are algebraically
additive. For a given protein characteristic, e.g., enzymatic
activity, urea
shifts the property in one direction
and methylamines
shift the property
in the opposite direction. Together,
at a 21 concentration
ratio, urea and
the methylamines
typically have no
net effect on protein characteristics.
Moreover, as long as the 21 concentration ratio is maintained,
the absolute concentrations
of urea and
methylamines
can be varied quite
widely without any diminution
in
their counteracting
effects (7).
One example of these counteracting effects is shown in Fig. 2, which
illustrates the influences of urea and
TMAO on the urea cycle enzyme,
argininosuccinate
lyase, isolated
from an elasmobranch
fish (thornback ray) and from the pig. Urea
inhibits
the enzyme noncompetitively; in the presence of 400 mM
urea the maximal
velocity (V,,,) is
decreased. TMAO at a concentration
of 200 mM increases the Vmax. When
400 mM urea and 200 mM TMAO
are both present in the in vitro assay
mixture, the observed Vmax is close
to the control (no urea or TMAO)
value. Effects of this sort have been
seen for several enzymes, and other
methylamines,
e.g., glycine betaine,
exhibit the same pattern of effect
shown here for TMAO.
In addition to illustrating
the effectiveness with which the methylamines can counteract urea pertur10
OH
I
I
H
concentrations
of urea we measured
in these same tissue samples. Thus,
as in the urea-rich fishes, the methylamine solutes of mammalian
kidney (which include at least glycine
betaine and glycerylphosphorylcholine) appear to be accumulated
to the
level at which maximal
counteraction of urea perturbation
is effected.
Mechanistic basis of urea and
methylamine effects on proteins
The contrasting effects of urea and
the methylamines
on protein structure probably derive from the different abilities of these solutes to penetrate the hydration
sphere around
proteins and to bind to proteins (3).
Solutes like urea that are able to
enter the hydration
layer and bind
to proteins favor protein unfolding.
Structure-stabilizing
solutes like the
methylamines
tend not to bind to
proteins because they are largely excluded from the water adjacent to
the protein surface. The tendency
for structure-stabilizing
solutes to be
excluded from the organized water
near the protein surface means that
a reduction in the amount of protein
surface area exposed to solvent (water) will be energetically
favored
when stabilizing solutes are present.
This structure-stabilizing
influence
follows directly from solution
entropy considerations.
Unfolding
of
the protein in the presence of structure-stabilizing
solutes would force
these solutes to partition into a reduced volume
of water, because
more of the water would now be
hydrating
the (increased)
protein
surface. This entropically
unfavorable distribution
of the structure-stabilizing solute molecules
is, therefore, the major factor responsible for
their stabilizing
effects on protein
structure.
The methylamine
solutes shown
in Fig. 1 contain groups that have
long been known to stabilize protein
structure. For example, quaternary
ammonium
ions are especially
strong structure stabilizers (3, 8). Although the effects of glycerylphosphorylcholine
on proteins have not
been examined,
its structural similarity to the other stabilizing
trimethylamine
solutes suggests that it,
too, is able to counteract urea perturbation of proteins.
Where else might the
counteracting solute strategy
be employed?
The discovery that the cells of the
renal papilla resemble in their osmotic characteristics
the cells of
urea-rich fishes is a striking example
of convergent evolution
at the biochemical level. The use of metabolic
waste products to build up high intracellular osmolarity-without
perturbing the structures and functions
of proteinsis an effective evolutionary strategy that would seem to
be accessible to many different types
of animals. In this evolutionary
context, it is interesting
to consider
other urea-rich organisms in which
the occurrence
of counteracting
methylamine
solutes could be important.
Several amphibians,
including the
African clawed frog, Xenopus laevis,
and the crab-eating frog, Rana cancrivoro, are known to accumulate
substantial concentrations
of urea in
their body fluids. It would be interesting to determine
whether these
species too have independently
“discovered” the counteracting
solute
strategy.
In other urea-rich organisms, the
accumulation
of urea-counteracting
solutes may not be advantageous. In
certain dormant stages of vertebrates
and invertebrates,
high concentrations of urea may build up, e.g., during estivation
of the African and
South American
lungfishes, desert
amphibians,
and desert snails (7). In
these organisms, urea may serve as
a metabolic depressant by inhibiting
protein function. The occurrence of
urea counteractants
would clearly
seem disadvantageous
in these dormant systems.
Portions
of this work
were supported
by
National
Science
Foundation
Grant
PCM8300983.
References
1. Balaban.
R. S., and M. A.
nuclear
magnetic
troscopy
of mammalian
Ph~siol. 245 (Cell Phqsiol.
gen-14
Knepper.
Nitroresonance
spectissues.
Am. 1.
14): C439-C444,
1983.
2. Beck, F., A. Dorge, R. Rick, and K. Thurau.
Intra- and extracellular
element
concentrations
of rat renal
papilla
in antidiuresis. Kidney
ht. 25: 397-403,
1984.
3. Low, P. S. Molecular
basis of the biological
compatibility
of Nature’s
osmolytes.
In:
Volume 1/February 1986
NIPS
11
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.2 on June 16, 2017
urea-rich
fishes suggests that, in
other organisms in which high urea
concentrations
are found, methylamine solutes may play a key role in
preserving the critical properties of
proteins. Recent findings
indeed
suggest that the osmotic gap and the
urea-perturbation
problems
facing
the cells of the renal papilla may be
solved by accumulating
methylamine solutes.
Initial
reports of glycerylphosphorylcholine
(Fig. 1) in dog kidney
(6) and glycine betaine in rat kidney
(5) have been followed by more detailed analyses of the concentrations
and qualitative
compositions
of
methylamines
in the mammalian
kidney. Balaban and Knepper (l), using nitrogen-14
nuclear
magnetic
resonance methods, reported that
trimethylamine
compounds
occurred at concentrations
of approximately 90 mmol/kg
fresh weight in
the inner medulla of the kidneys of
the rat and the. rabbit. Because this
concentration was based on total tissue mass, the estimated intracellular
concentration of the trimethylamine
solutes was suggested by these authors to be near 200 mmol/kg
intracellular water. They could not detect
the trimethylamines
in urine, which
implied that these compounds were
found exclusively in the intracellular compartment
of the inner medulla. About 20% of the methylamines were accounted for by glycerylphosphorylcholine;
the remaining
fraction was not identified.
We recently have used high-performance
liquid
chromatography
methods to examine
further
the
methylamines
present in kidney
(cortex, outer medulla,
and inner
medulla) of the dog (Finley and Somero, unpublished
data). As in the
Balaban and Knepper study (l), we
found no methylamines
in blood serum or urine, but we detected glytine betaine at concentrations
up to
approximately
90 mmol/kg
intracellular water in the inner medulla region. No TMAO could be detected.
Glycine betaine concentrations
increased from the cortex to the inner
medulla in concert with rising total
osmolarity
and urea concentration
of the different tissue regions.
Our estimates of the intracellular
concentrations
of glycine betaine in
these three kidney
regions were
roughly one-third
to one-half the
Proceedings
of the First
International
Congress on Comparative
Physiology
and
Biochemistry,
edited by R. Gilles. Berlin:
Springer-Verlag.
In press.
4. Macmillen,
R. E., and A. K. Lee. Australian desert mice: independence
of exogenous water. Science Wash. DC 158: 383385,
1967.
D. Bowlus,
and G. N. Somero. Living with
water stress. Science Wash. DC 217: 1214-
5. Martin,
J. J., and J. D. Finkelstein.
Enzymatic determination
of betaine
in rat tissues. Anal. Biochem.
111: 72-76,
1981.
6. Schimassek,
H., D. Kohl, and T. Bucher.
Glycerylphosphorycholin,
die
Nierensubstanz
“Ma-Mark”
von Ullrich.
Biothem. Z. 331: 87-97,
1959.
7. Yancey,
P. H., M. E. Clark,
S. C. Hand, R.
1222,
8.
213,
Putrescine, Spermidine,
and Spermine
ported to the newly formed Royal
Society of London in the famous letter in which he first described spermatozoa.
The name spermine was given to
the base in 1888 by Ladenburg and
Abel, but its chemical
nature remained
unclear
until
the EJZOs,
when Rosenheim and his collaborators finally confirmed the structure
by synthesis. In addition, they discovered a related base, which they
named spermidine.
In 1885 two
other related bases were isolated
from decomposing
animal material
by Brieger, and in 1886 Ladenburg
confirmed
their structures by synthesis. They were named putrescine
and cadaverine because of their origin and foul smell of putrefaction.
3OO-Year-old history
Olle Heby is Associate
Professor
in the Department
of Zoophysiology,
University
of Lund,
Helgonavagen
3, S-223 62 Lund, Sweden.
12
NIPS
Volume 1/February 1986
Biosynthetic
Occurrence
Ornithine
The polyamines
are found not
only in semen and in decomposing
animal material-they
are ubiquitous in nature. Putrescine and spermidine are synthesized in all cells,
but spermine
only in eukaryotic
cells. Viruses
and physiological
fluids also contain polyamines,
but
these are derived from cells.
pathway
decarboxylase
NH2(CH2)4NH2
(ODC) is
putrescine
co&wine
%2)5NH2
spw midine
NH2(cH#@‘-“$bNH2
fw2ct$p(CH24NH(CH2)3NH2 spermine
FIGURE 1. Chemical structures for the naturally occurring polyamines.
0886
17 14/86 $1.50 0 1986 Int Union
Physiol.
Sci/Am
Physlol.
Sot
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At a physiological
pH, putrescine,
spermidine,
and spermine are protonated and possess two, three, and
four positive charges, respectively
(cf. Fig. 1). The positive charge distribution
in the spermine molecule
makes it bind strongly to the negatively charged phosphate groups in
double-helical
regions of nucleic
acids. Spermine
molecules occupy
the small groove and neutralize two
phosphate
groups in each strand,
thus stabilizing
the double helix by
binding its two strands together.
Moreover, spermine is by far the
most potent inducer of the transition
from the usual right-handed
(BDNA) to a left-handed (Z-DNA) double-helical
conformation
of DNA.
The Z-DNA form is favored by sequences with alternating
purines
and pyrimidines,
especially alternating guanine and cytosine residues.
Potential Z-DNA forming sequences
are highly repeated in the human
genome. The actual presence of the
left-handed
DNA conformation
is
evident from the finding that antibodies to Z-DNA occur in patients
with lupus erythematosus.
Z-DNA
sequences may be important in mutagenesis in recombination
and in
the control of gene expression, and
spermine may play the role of an
conformational
inducer
of the
switch.
Putrescine
was first isolated from putrifying
meat and was thought of
as a decomposition
product; spermine
was named from its occurrence
in semen. These polyamines,
however, are now known to have
important
roles in cell growth and differentiation.
Their physiological
significance
can be studied by analyzing
the consequences
of
depletion
of the cellular
polyamine
content. The results include arrest
of cell growth, differentiation,
and division.
Further results suggest
that blocking
of polyamine
synthesis may have broad implications
in
clinical
medicine.
The history of the polyamines began in 1678 with the discovery by
Leeuwenhoek
of the crystallization
of spermine from human semen. The
observation
was made with his
primitive
microscope
and was re-
1980.
Properties
Olle Heby
The general misconception
that the
polyamines
putrescine, spermidine,
and spermine
(Fig. 1) are cellular
degradation products initially
hampered research within
this field.
During the last decade, however,
polyamine
research has truly burgeoned, and as a result many important roles have been discovered. In
fact, it now appears that cells depend
on polyamines for many of their life
processes. Due to the multitude
of
new findings, I am compelled
to
limit the present discussion. The detailed aspects of polyamine
biochemistry
and physiology
may be
found in more comprehensive
reviews and in proceedings of recent
meetings (l-3, 5, 6).
1982.
Yancey,
P. H., and G. N. Somero.
Methylamine
osmoregulatory
solutes
of elasmobranch
fishes counteract
urea inhibition of enzymes.
I. Exp. Zool. 212: 205-