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JEZ 767
THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:115–118 (1997)
RAPID COMMUNICATIONS
Tissue-Specific Forms of b-Hydroxybutyrate
Dehydrogenase Oxidize the D- or L-Enantiomers of
b-Hydroxybutyrate in the Terrestrial Gastropod
Cepaea nemoralis
J.A. STUART AND J.S. BALLANTYNE*
University of Guelph, Guelph, Ontario N1G 2W1, Canada
β-hydroxybutyrate dehydrogenase (BHBDH)
catalyzes the interconversion of the ketone bodies acetoacetate (Acac) and D-b-hydroxybutyrate
(BHB). In virtually all animals, including mammals and fish (Newsholme and Leech, ’83) and
freshwater molluscs (Meyer et al., ’86), BHBDH
exists within the mitochondria. The mammalian
BHBDH is a popular model for the study of the
kinetics of membrane-bound enzymes since it has
an obligate requirement for certain phospholipid
species (Isaacson et al., ’79).
We have recently described a different and
unique form of BHBDH found in the terrestrial
snail Cepaea nemoralis (Stuart and Ballantyne,
’96) which differs in two respects from the enzyme
described above. This enzyme occurs in the cytosol
of hepatopancreas cells and oxidizes exclusively the
L-enantiomer of BHB. This form of BHBDH does
not occur in all tissues of C. nemoralis. Here, we
describe the presence in other tissues of another
cytosolic form of BHBDH with an enantiomeric
specificity for the D-stereoisomer and demonstrate
that it is a separate protein from L-BHBDH.
MATERIALS AND METHODS
C. nemoralis were collected from fields near the
University of Guelph campus. A colony of these
snails was maintained in the laboratory, in terrariums kept near 30% ambient humidity with
periodically moistened sphagnum moss. Snails
were fed lettuce ad libitum.
Tissue preparation for enzyme assays was essentially as described in Stuart and Ballantyne
(1996). Active (withdrawn from shell) adult snails,
approximately 1.5 cm diameter, were decapitated
and deshelled.
Tissues were prepared for enzyme assays by
placing the excised tissue in 2 ml of ice-cold mitochondrial isolation medium (20 mM N-[2© 1997 WILEY-LISS, INC.
hydroxyethyl]piperazine-N´-[2-ethanesulfonic
acid] [HEPES] and 100 mM sucrose, pH 7.5) and
homogenizing by five passes of a Potter-Elvejhem
homogenizer with a teflon pestle attached to a drill
press operated at <100 revolutions per minute.
All subsequent procedures were carried out at 5°C.
Two separate centrifugation protocols were applied
to homogenates. Initially, we used a 10,000g, 10
min centrifugation to separate tissues into mitochondrial and cytosolic fractions and verified the
absence of BHBDH activity in the 10,000g pellet.
Subsequently, homogenates were centrifuged at
200g for 10 min and the resultant pellet discarded.
This supernatant was centrifuged at 10,000g for
10 min and the resultant supernatants decanted.
The remaining mitochondrial pellets were resuspended in 2 ml of mitochondrial isolation medium.
The supernatant was centrifuged at 30,000g for
10 min and the pellet discarded, and the resultant supernatant was considered to be the cytosolic fraction. Both tissue fractions were then
sonicated with a 15 sec burst at 80% output, 50
W, on a Vibra-Cell sonicator (Sonics & Materials
Inc., Danbury, CT). Aliquots of these fractions
were used directly in enzyme assays.
Citrate synthase (CS) and BHBDH activities
were measured as described by Stuart and Ballantyne (1996), with the following exception: the
BHBDH assay medium contained 2 mM NAD
and either 400 mM DL-BHB or 200 mM D- or LBHB in 50 mM imidazole, pH 8.0. All chemicals for enzyme assays were purchased from
Sigma (St. Louis, MO) and were of the highest
purity available.
Tissue were prepared for electrophoresis on cel*Correspondence to: J.S. Ballantyne, Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Received 17 October, 1996; Revision accepted 3 December 1996
116
J.A. STUART AND J.S. BALLANTYNE
lulose-acetate gels by adding about 20 mg of tissue to 200 µl (heart), 100 µl (kidney), and 250 µl
(hepatopancreas) of tris-glycine gel buffer (25 mM
Tris, 200 mM glycine, pH 9.0) in eppendorff tubes
and homogenizing with a tight-fitting plastic pestle.
Homogenates were clarified with a 5 min, 10,000g
centrifugation. A sample of each supernatant was
applied using a Super Z Applicator (Helena Laboratories, Beaumont, TX) to a 76 × 76 mm Titan III
cellulose-acetate plate (Helena Laboratories), which
had been presoaked in gel buffer.
Gels were placed in a plexiglass electrophoresis
tank, with gel buffer used as electrode buffer. A 300
V differential was applied, using a Heathkit Regulated Power Supply (model IP- 2717A) (Phipps &
Bird, Inc., Richmond, VA), for 2 hr at 5°C. Gels were
removed and stained with a solution containing 0.3
mg MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), 0.07 mg phenazine
methosulfate, 1.0 mg NAD, and 33.6 mg DL- or Dor L-BHB in 3 ml tris-glycine (pH 9.0) added to 2
ml agarose gel maintained at 60°C. The staining
solution was removed from gels after approximately 1 hr, and gels were photographed while
still moist.
with cytosolic enzyme was 3.4%, 1.0%, and 1.4%
of total BHBDH activity in ventricle, kidney, and
hepatopancreas, respectively.
Homogenates of ventricular tissue oxidize almost exclusively D-BHB, whereas the hepatopancreas is specific for L-BHB and the kidney oxidizes
both stereoisomers (Table 1). Some apparent nonspecificity of BHBDH activity in ventricle and
hepatopancreas was likely the result of small
amounts of the other stereoisomer present as an
impurity in the commercial BHB preparations.
Oxidation of L-BHB in the ventricle occurred at
0.5% the rate of D-BHB oxidation. The commercial
L-BHB preparation was 97% pure and contained
approximately 0.4% D-BHB. This contamination
likely accounts for the low activity of L-BHBDH
in the heart. Similarly, oxidation of D-BHB in the
hepatopancreas occurred at 4.5% the rate of LBHB oxidation. The commercial D-BHB preparation was 98% pure, with less than 2% L-BHB
contamination. Thus, contaminating L-BHB likely
accounts for the low rate of oxidation of D-BHB
in this tissue.
The oxidation of D-BHB by D-BHBDH appears
to be inhibited by L-BHB. In the kidney, activity
with DL -BHB as substrate is less than would be
expected by summing the independent D and L activities (Table 1). Similarly, BHBDH activity in
the ventricle is greatly decreased when assayed
with DL-BHB compared to D-BHB.
Staining of electrophoretic gels with DL -BHB
clearly demonstrates that two separate enzymes
are involved in the oxidation of the enantiomers
of BHB in the tissues examined. Two distinct
bands which migrate different distances from the
origin are visible (Fig. 1). The ventricle band (DBHBDH) occurs equidistant from the origin to the
top kidney band, indicating that this form occurs
in both tissues. Similarly, the hepatopancreas
band ( L-BHBDH) matches the bottom kidney
band. The kidney thus contains both forms of the
enzyme ( L-BHBDH and D-BHBDH). Separate
staining with D- or L -BHB corroborates the
RESULTS
We used CS as an indicator of the extent of leakage of mitochondrial matrix enzymes from mitochondria damaged in the tissue fractionation
process (Table 1). The appearance of CS in the
cytosolic fractions of kidney and hepatopancreas
is 11% and 13% of summed mitochondrial and cytosolic activities, respectively, indicating minimal
leakage from the mitochondrial matrices in these
tissues. In the ventricle, cytosolic CS accounted
for 45% of total activity, suggesting greater
intramitochondrial leakage in this tissue. This
greater proportion of damaged mitochondria in the
fractionation of ventricle tissue did not affect the
interpretation of results. Virtually no mitochondrial BHBDH activity was detected in any tissue.
Cross-contamination of the mitochondrial fraction
TABLE 1. Activities of citrate synthase (CS) and β-hydroxybutyrate dehydrogenase (BHBDH) in mitochondrial and cystosolic
compartments of Cepaea nemoralis tissues (n = 5)
Enzyme(s)
Compartment
CS
CS
DL-BHBDH
DL-BHBDH
D-BHBDH
L-BHBDH
Mitochondria
Cytoplasm
Mitochondria
Cytoplasm
Cytoplasm
Cytoplasm
Ventricle
2.82
3.40
0.35
10.12
36.36
0.17
±
±
±
±
±
±
0.34
0.90
0.22
1.33
4.55
0.08
Kidney
1.87
0.23
0.03
2.79
3.99
1.61
±
±
±
±
±
±
0.55
0.07
0.02
0.37
0.65
0.31
Hepatopancreas
1.19
0.18
0.02
1.30
0.06
1.37
±
±
±
±
±
±
0.29
0.06
0.01
0.24
0.02
0.25
STEREOSPECIFIC b-HYDROXYBUTYRATE DEHYDROGENASES
Fig. 1. Cellulose acetate gel of ventricle (lane 1), hepatopancreas (lane 2), and kidney (lane 3) stained with DL -BHB
(see text).
identification of exclusively L -BHBDH in the
hepatopancreas and exclusively D-BHBDH in the
ventricle (not shown).
DISCUSSION
Our results indicate that two distinct forms of
BHBDH exist in C. nemoralis tissues. Both enzymes are cytosolic, and each is specific for one
stereoisomer of BHB.
Subcellular location of BHBDH in C.
nemoralis tissues
There is no evidence for the existence of the typical mitochondrial membrane-bound form of BHBDH
in heart, kidney, or hepatopancreas of the terrestrial snail C. nemoralis. In all of these tissues,
BHBDH is localized to the cytosol. Very low activities of the enzyme in the mitochondrial compartments of these tissues are consistent with
minor contamination from the cytosol during the
tissue fractionation procedure. The subcellular
location of Cepaea BHBDH contrasts with that of
freshwater gastropods, where BHBDH is mitochondrial (Meyer et al., ’86, personal observations).
Stereospecificity of BHBDH
C. nemoralis have the ability to utilize both
enantiomers of BHB, each catalyzed by a different protein, as indicated by cellulose acetate
electrophoresis. While the ventricle and the
hepatopancreas oxidize exclusively D-BHB or LBHB, respectively, the kidney can oxidize both
substrates. The demonstration of two separate
proteins involved in the utilization of D- and L-
117
BHB rules out another possible mechanism which
could account for the pattern of L- and D-BHB utilization observed in Table 1. Racemization of one
enantiomer to another, e.g., L-BHB to D-BHB,
could precede oxidation by D-BHBDH. This would
require two separate enzymes, a racemase and a
BHBDH. However, these proteins would likely
have been separated by the gel matrix. Thus,
where L- or D-BHB was directly oxidized, a single
clear band would have been visible. Where racemization was coupled to oxidation, a blurred band
would have occurred equidistant from the origin
to the clear band, indicating that both the racemase and dehydrogenase had migrated similar
distances. Alternatively, if the racemase and dehydrogenase migrated very different distances, no
band would have been visible. Thus, as two distinct bands were clearly visible at different distances from the origin, two separate enzymes
must be responsible for the oxidation of the stereoisomers of BHB.
Although many enzymes exist as multiple isoforms, with individual isozymes often localized to
specific tissues, these isoforms typically differ from
one another in their kinetic properties, including
Michaelis constants and sensitivity to cofactors. The
differential occurrence, within tissues of a single
organism, of two distinct isoforms of an enzyme
which are specific for different enantiomers of a substrate is, to our knowledge, unique to this terrestrial snail. We are not aware of other examples of
this phenomenon in the animal kingdom.
Lactate dehydrogenase (LDH) occurs as either
D-LDH or L-LDH throughout the animal kingdom
(Long, ’76). However, no organism has been shown
to have both D- and L-LDH (Long, ’76). Some molluscs use both the D- and L-enantiomers of certain
amino acids (Ballantyne and Chamberlin, ’94). Alanine, in particular, occurs as both D- and L-stereoisomers in high concentrations in tissues of some
marine bivalves (Yamada and Matsushima, ’92). In
these molluscs, however, there is no evidence of a
D- and L-alanine aminotransferase (Hayashi, ’93).
Instead, a D-amino acid oxidase or a racemase
have been implicated in the oxidation of intracellular D-alanine (Matsushima and Hayashi,
’92; Ballantyne and Chamberlin, ’94). These authors suggest that this may be significant in maintaining a role for D-alanine as an intracellular
osmolyte, while L-alanine is accessible to the oxidizable substrate pool.
Although the mechanism of D- and L-BHB metabolism in Cepaea differs from that of D- and Lalanine in some bivalve molluscs, the designs are
118
J.A. STUART AND J.S. BALLANTYNE
similar in that in both cases a substrate is made
unavailable for oxidation by a tissue. In Cepaea,
hemolymph L-BHB could not serve as an energy
substrate for the ventricle nor could D-BHB be
used by the hepatopancreas. This effectively creates a partitioning of BHB. Such a design would
allow BHB to be directed to specific tissues or to
specific cell types within a tissue, e.g., kidney,
where D-BHBDH and L-BHBDH could occur in different cell types.
Achieving this extra level of control over the
metabolism of BHB may be an important adaptation of terrestrial snails, where relatively high activities of BHBDH and other enzymes of ketone
body metabolism suggest that ketone bodies play
a prominent role in energy metabolism (Stuart
and Ballantyne, ’97). As fatty acids do not appear
to be oxidized substantially by peripheral tissues
(Stuart and Ballantyne, ’97), ketone bodies may
be an important means of distributing lipid carbon from central stores to peripheral tissues.
Thus, the controlled synthesis of one or the other
enantiomer of BHB may allow stored lipid carbon to be directed to a certain tissue or tissues
for oxidation.
In summary, terrestrial snails possess a unique
organization of ketone body metabolism, which differs from the mammalian model in the subcellular compartmentation and stereospecificity of
BHBDH. The present study reports the first demonstration of a cytosolic D-BHBDH in animal tissues. Similarly, the presence in different tissues
of two distinct forms of BHBDH, each specific for
a single enantiomer of BHB, is, we believe, unique.
This organization of ketone body metabolism results in a partitioning of BHB availability between
tissues, and the physiological implications of this
phenomenon should be investigated. As both Dand L-BHBDH are present in substantial activities, it is likely that they play important roles in
the intermediary metabolism of terrestrial mollusc tissues.
Finally, this description of BHBDH isoforms in
C. nemoralis tissues should be noted by popula-
tion geneticists as gel staining with DL-BHBDH
could give misleading results if not interpreted
on the basis outlined above.
ACKNOWLEDGMENTS
We thank the people of Paul Hebert’s lab for
their assistance with gel electrophoresis. This
study was supported by a Natural Sciences and
Engineering Research Council (NSERC) operating grant to J.S.B.
LITERATURE CITED
Ballantyne, J.S., and M.E. Chamberlin (1994) Regulation of
cellular amino acid levels. In: Cellular and Molecular Physiology of Cell Volume Regulation. Kevin Strange, ed. CRC
Press, Ann Arbor, MI, pp. 111–122.
Hayashi, Y.S. (1993) Alanine aminotransferase from gill tissue of the brackish-water bivalve Coribula japonica (Prime):
Subcellular localization and some enzymatic properties. J.
Exp. Mar. Biol. Ecol., 170:45–54.
Isaacson, Y.A., P.W. Deroo, A.F. Rosenthal, R. Bittman, J.O.
McIntyre, H.-G. Bock, P. Gazzotti, and S. Fleischer (1979)
The structural specificity of lecithin for activation of purified D-β-hydroxybutyrate apodehydrogenase. J. Biol. Chem.,
254:117–126.
Long, G.L. (1976) The stereospecific distribution and evolutionary significance of invertebrate lactate dehydrogenases.
Comp. Biochem. Physiol., 55B:77–83.
Matsushima, O., and Y.S. Hayashi (1992) Metabolism of Dand L-alanine and regulation of intracellular free amino acid
levels during salinity stress in a brackish-water bivalve Corbicula japonica. Comp. Biochem. Physiol. A, 102:465–471.
Meyer, R.W., W. Becker, and M. Klimkewitz (1986) Investigations on the ketone body metabolism in Biomphalaria
glabrata: Influence of starvation and of infection with Schistosoma mansoni. J. Comp. Physiol., 156:563–571.
Newsholme, E.A., and A.R. Leech (1983) Biochemistry for
the Medical Sciences. John Wiley & Sons, New York.
Stuart, J.A., and J.S. Ballantyne (1996) Subcellular organization of intermediary metabolism in the hepatopancreas of the terrestrial snail, Cepaea nemoralis: A
cytosolic β-hydroxybutyrate dehydrogenase. J. Exp. Zoo.,
274:291–299.
Stuart, J.A., and J.S. Ballantyne (1997) Importance of ketone bodies to the intermediary metabolism of the terrestrial snail, Archachatina ventricosa: Evidence from enzyme
activities. Comp. Biochem. Physiol. (in press).
Yamada, A., and O. Matsushima (1992) The relation of Dalanine and alanine racemate activity in molluscs. Comp.
Biochem. Physiol. [B], 103:617–621.
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