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 Active (withdrawn from shell) adult snails, approximately 1.5 cm diameter, were decapitated and deshelled.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. 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