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The ontogeny of fructose diphosphate aldolase B in the chick
DEVELOPMENTAL
BIOLOGY
The Ontogeny
27, 143-149 (1%‘)
of Fructose
Diphosphate
Aldolase
I3 in the
Chick
HERBERT G. LEBHERZ Laboratory
for Developmental
Biology,
Zoology Institute, Zurich, Switzerland
Accepted
Swiss Fedeml Institute
of Technology,
September 27, 1971
The three homologous parental fructose-l, 6-diphosphate aldolases A, B, and C have previously been demonstrated in vertebrates. Although five-membered A-B, A-C, and B-C hybrid sets are readily produced in vitro by reversible dissociation of the appropriate parental enzymes, only A-B and A-C sets have been found heretofore in vertebrate tissues. In the present studies, cellulose-polyacetate electrophoresis followed by specific enzymatic staining has demonstrated the existence of B-C hybrids in uiuo. B-C hybrids were found in early embryonic chick liver (51 to 8h days) and in yolk sac membranes. The aldolase activity profiles suggest a transition in synthesis from aldolase C to predominantlyaldolise B subunits during early chick liver embryogenesis. The fact that B-C hybrids were detected suggests that the C to B transition takes place within individual cells and is not due to the preferential proliferation of cells containing aldolase B. The timing of the C to B transition in liver is consistent with the proposed role of aldolase B in gluconeogenesis. Finally, the present studies are discussed in relation to other aldolase transitions observed to be associated with vertebrate tissue embryogenesis and with certain disease states.
Subunits of aldolases A, B, and C can interact to form hybrid molecules. Since these aldolases are composed of four subunits (Kawahara and Tanford, 1966; Penhoet et al., 1967), five-membered hybrid sets are formed by the binary combination of the parental subunits into tetramers. Five-membered hybrid sets are readily produced in vitro by reversible acid dissociation of the appropriate parental enzymes (Penhoet et al., 1966) and are observed in tissues in which two of the parental enzymes are synthesized simultaneously (Lebherz and Rutter, 1969). Although A-B, A-C, and B-C hybrid sets are readily produced in vitro (Penhoet et al., 1966) only A-B and A-C sets have been observed in vivo. B-C hybrids have never been detected in animal tissues even though certain organs (i.e., chicken kidney) contain both the A-B and A-C sets (Lebherz and Rutter, 1969). The distinctive catalytic properties of aldolases A and B suggest that, in vivo, these enzymes have different physiological functions. Aldolase A appears to be tailored for a glycolytic role while aldolase B
INTRODUCTION
The three parental fructose-l, 6-diphosphate (FDP) aldolases were first isolated from mammalian tissues: aldolase A, the classical muscle enzyme (Taylor et al., 1948); aldolase B, from liver (Rajkumar et al., 1967); and, aldolase C, from brain (Penhoet et al., 1966, 1969a). Although these enzymes are similar in many respects, they can be distinguished on the basis of their catalytic, molecular, electrophoretic, and immunological properties (Rutter et al., 1963b, 1968; Penhoet et al., 1969b). Homologs of the rabbit A, B, and C enzymes have more recently been isolated from chicken tissues and appear to be quite similar to the corresponding rabbit aldolases (Marquardt, 1969, 1970; 1971a, b). In addition, extensive phylogenetic studies have demonstrated the presence of the three parental enzymes, and hence their structural genes, in a wide variety of vertebrates (Lebherz and Rutter, 1969). It seems likely, therefore, that aldolases A, B, and C are present in all vertebrate organisms. 143 Copyright
0 1972 by Academic
Press, Inc.
144
DEVELOPMENTAL BIOLOGY
may function mainly in gluconeogenesis and fructuse metabolism (Rutter et al., 1963a). Both liver and kidney cortex are rich in gluconeogenic activity and the metabolism of fructose via fructose l-phosphate (F-l-P) occurs primarily in liver (Benoy and Elliott, 1937; Krebs et al., 1963). The observed restricted tissue distribution of aldolase B to vertebrate liver and kidney (Lebherz and Rutter, 1969) and the observed lack of functional aldolase B in patients suffering from fructose intolerance disease (Nordmann et al., 1968) are in accordance with the proposed physiological functions of aldolase B. There is yet no known catalytic basis to suggest that aldolases A and C have distinct physiological functions within the cell (Penhoet et al., 196913). The observations that the early chick embryo contains predominantly aldolase C while aldolase B is the predominant form found in adult liver (Lebherz and Rutter, 1969) suggest that a transition in synthesis from aldolase C to B subunits and, hence, the generation of B-C hybrids may occur during chick liver embryogenesis. The present studies demonstrate the presence of B-C hybrid aldolases in early embryonic chick liver (5-8 days) and in yolk sac membranes. In addition, the data suggest that a transition in synthesis from aldolase C to predominantly aldolase B subunits takes place during early chick liver embryogenesis. Finally, the timing of the C to B transition is consistent with the proposed role of aldolase B in gluconeogenesis. MATERIALS
AND
METHODS
Purified enzymes, coenzymes, substrates, and histochemical reagents were obtained from either the Sigma Chemical Co., St. Louis, Missouri, or Calbiochem Inc., Los Angeles, California. Materials for electrophoresis including Sepraphore III cellulose-polyacetate strips were purchased from the Gelman Instrument Co.,
VOLUME 27, 1972
Ann Arbor, Michigan. All other chemicals were of reagent grade. Embryonic chick tissues were obtained from eggs incubated at 38.2”C for appropriate times. Tissues were homogenized at 4°C in approximately 2 volumes of 0.06 M sodium barbital, 0.06% 2-mercaptoethanol, pH 8.6, either in depression wells with a glass rod or in a glass-glass hand homogenizer. Because of their small volumes, homogenates of early embryonic tissues were generally used without further treatment, but care was taken to omit particulate matter from the samples. All yolk sac membrane homogenates were centrifuged at 17,500 g (2-4°C) for 50 min and the supernatants were used for study. Determination of aldolase activity using either 2.5 mM FDP or 10 mM F-l-P as substrate was made by the spectrophotometric method of Blostein and Rutter (1963) in 0.06 M Tris HCl, pH 7.6 at 25°C. A unit of activity is expressed as the cleavage of 1 pmole of substrate per minute per milliter of extract. Cellulose-polyacetate electrophoresis was performed in 0.06 M sodium barbital, 0.06% 2-mercaptoethanol, pH 8.6 according to the method of Penhoet et al. (1966). Tissue homogenate, 2-5 ~1 containing 0.001 to 0.005 unit of aldolase activity, was applied to the strips and electrophoresis was performed at 250 V for 90 min. The strips were then stained for FDP cleavage activity as described by Penhoet et al. (1966). Location of F-l-P cleavage activity was determined by incubating strips with staining gels containing 0.02 M F-l-P and 0.1 mg/ml ol-glycerolphosphate dehydrogenase-triose phosphate isomerase in place of the 0.01 M FDP used above. The strips and staining gels were incubated at 37°C until good color development (Formazan production) occurred. RESULTS
The parental aldolases A, B, and C from those from most vertebrates, including
LEBHERZ
Aldolase
chicken, have different electrophoretic mobilities (Lebherz and Rutter, 1969). In the present studies, the parental and hybrid aldolases were resolved by cellulosepolyacetate electrophoresis and located by specific enzymatic staining. Positive identification of the activities was made on the basis of electrophoretic mobility and substrate specificity. Substrate specificity is particularly useful in distinguishing aldolase B activity from aldolase A and C activity. Aldolase B has a considerably lower K, for fructose l-phosphate (F-l-P) than do aldolases A and C. Furthermore, aldolase B functions with the same efficiency with either FDP or F-l-P as substrate (FDP/F-1-P = 1). In contrast, aldolases A and C function more efficiently with FDP and have FDP/F-1-P activity ratios of 10 or greater (Rutter et al., 1968; Penhoet et al., 1969b). The relatively high efficiency of F-l-P cleavage exhibited by aldolase B is presumably a reflection of this enzyme’s function in the metabolism of fructose via fructose lphosphate (Rutter et al., 1963a) and, therefore, is a suitable diagnostic parameter for aldolase B activity. Since the catalytic properties of aldolase subunits are apparently the same whether they exist in homomeric or heteromeric (hybrid) combinations (Penhoet and Rutter, 1971), the relative intensities of aldolase activities stained with FDP and F-l-P can be used to positively identify those tetramers which contain B subunits. Figure 1 demonstrates the resolution and identification of aldolase activities in 17-day-chick liver and skeletal muscle. When the strips are stained with FDP (top) the entire A-C hybrid set (A,, A,C, A2C,, AC,, C,) is readily apparent in the muscle profile while aldolase B, which migrates more cathodally than the other activities, is the predominant enzyme found in liver. In contrast, when similar strips are stained with F-l-P, only aldolase B of liver is developed (bottom). The FDP and F-l-P activity profiles of
B in Chick Embryo
145
FIG. 1. Aldolase activity profiles of 17.day chick leg skeletal muscle (M) and liver (L) using fructose1,6-disphosphate (FDP) (top) and fructose l-phosphate (F-1-P) (bottom) as substrates. See Materials and Methods section for details. Muscle and liver extracts were electrophoresed on the same strips.
liver and heart from early chick embryos are presented in Fig. 2. The 17-day muscle profiles are included for comparison. All heart samples contained the A-C activities Cd, ACB, and A&,. These same activities were observed in total homogenates of 3-day embryos. The transition from predominantly aldolase C to the entire A-C set which is present in adult heart (Lebherz and Rutter, 1969) takes place after 17 days of incubation, and probably occurs mainly after hatching. Notice that the activities of muscle and heart are not revealed with F-l-P. The two most anodal activities detected in liver homogenates correspond to the enzymes C, and AC, (Fig. 2). The profiles of 4-day liver are essentially identical to those of heart and no aldolase B activity was detected in 4-day liver by staining with F-l-P. In contrast, major activities which do not correspond to members of the A-C set were detected in 5day liver. The three prominent cathodal activities have mobilities expected for the B-C hybrids BC,, B,C,, and B,C. Low levels of the homotetramer B,, were also detected with FDP. Positive identification of these activities as members of the B-C set was made from the F-l-P profiles. As shown, activities corresponding to B,, B,C, B2C2, and BC, were readily apparent.
146
DEVELOPMENTAL BIOL~CY
VOLUME 21, 1972
FDP Q +
FIG. 2. Aldolase activity profiles of embryonic chick tissues (M = muscle, H = heart, I, = liver). All heart and liver extracts of each age were electrophoresed on the same strips. The slight staining at the origin of the 7 day fructose l-phosphate (F-1-P) profile is presumably due to enzyme activity associated with particulate matter. See Materials and Methods section for details.
The 6-day liver contains predominantly aldolases B,, B,C, and B,C,, although B,C, was only weakly detected in the F-l-P profile. The transition toward aldolase B continues during day 7 and is essentially completed by day 8 as suggested by the F-l-P profiles. Aldolase B, was the only major activity detected in the 17-day liver. Very few activities corresponding to A-B or A-B-C hybrids were detected
during the C to B transition. On occasion, the C to B transition was observed to be completed slightly later than the sample shown in Fig. 2. However, in all cases the transition was essentially completed by day 10, as evidenced by activity profiles and the similarity between the FDP/F-lP activity rations of IO-day and adult liver samples (FDP/F-1-P = 1.2-1.3). It is essential to point out that the FDP
LEBHERZ
Aldolase
and F-l-P activity profiles presented above reflect to a great extent the catalytic behavior of the three aldolase subunits. The increased F-l-P staining of the B-C hybrids toward the B terminus is inof fluenced by the “specific activities” B subunits in the various tetramers. For example, the hybrid B,C, exhibits one half of the B activity per tetramer as does the enzyme B,. Conversely, the relative intensities of the activities revealed with FDP deceptively emphasize the molar concentrations of tetramers containing A and C subunits since aldolases A and C have considerably higher FDP turnover numbers than does aldolase B (Penhoet et al., 1969b). Since many of the biochemical activities found in liver are also present in yolk sac membrane (YSM) and since the YSM presumably functions in a liver capacity during early chick development (Croisille and Le Douarin, 1965), it was of interest to determine which aldolases are present in this membrane during early development. Studies similar to those performed on chick liver revealed the presence of aldolase B and B-C hybrids in the YSM of all embryos tested (3-10 days). No aldolase B activity was detected in another membrane, the allantois, embryonic which instead contained the A-C activities Cd, ACB, and A&,. DISCUSSION
The present studies demonstrate for the first time the existence of B-C hybrid aldolases in vivo. These hybrids were detected in early embryonic chick liver (5-8 days) and in yolk sac membranes. The fact that B-C hybrids exist in these tissues suggests that subunits of aldolases B and C are simultaneously synthesized within the same cells. In contrast, the observed lack of B-C hybrids in adult vertebrate organs (Lebherz and Rutter, 1969) indicates that, in differentiated tissues, aldolase B and C subunits are rarely, if ever, synthesized within the same cells at the same time.
B in Chick Embryo
147
Whether or not there is any physiological or other basis for the apparent lack of B-C hybrids in differentiated vertebrate organs is not known. The low levels of A-C activities detected in embryonic and adult liver are presumably located in erythroid or other cells (Rutter et al., 1963a; Rutter and Weber, 1965). The transition from aldolase C to predominatly aldolase B observed between approximately 5 and 8 days of incubation as well as the observed decrease in FDP/F-1-P activity ratio from 17 to 2 during this time (Weber, 1965) presumably reflect a transition in synthesis from predominantly aldolase C to predominantly aldolase B subunits by the early embryonic chick liver. This interpretation is supported by the studies of Weber (1965) and Weber and Rutter (1964) which demonstrated a preferential synthesis of aldolase B subunits during the A to B transition associated with rat liver embryogenesis. other interpretations of the However, one based on present data, including preferential degradation of C over B subunits, cannot be eliminated at this time. The fact that B-C hybrids exist supports the contention that the C to B transition observed occurs within individual cells, presumably parenchymal, and is not due to the preferential proliferation of cells containing aldolase B. The finding of appreciable aldolase B activity in yolk sac membrane is also of interest from a physiological point of view. The YSM and liver possess many biochemical activities in common; moreover, this embryonic membrane appears to carry out liver functions prior to the time liver has the capacity to do so (Willier, 1968; Croisille and Le Douarin, 1965). It is perhaps significant, therefore, that aldolase B activity has been detected in YSM several days prior to the time this enzyme appears in liver. The timing of the C to B transition in chick liver is consistent with the gluconeogenie function assigned to aldolase B. The
148
DEVELOPMENTAL BIOLOGY
transition toward aldolase B is completed just prior to the time of marked gluconeogenie activity exhibited by embryonic chick liver between approximately 11 and 15 days of incubation (Ballard and Oliver, 1963). In addition, the A to B transition observed during rat liver embryogenesis, 14 days to birth (Rutter and Weber, 1965; Weber, 1965), is completed just prior to the onset of gluconeogenesis in rat liver observed to occur at birth (Ballard and Oliver, 1963). Therefore, in both organisms high levels of aldolase B are present in liver at the time when appreciable gluconeogenie activity becomes apparent. The present demonstration of a C to B transition during early chick liver embryogenesis supplements earlier studies on the aldolases of developing vertebrate tissues (Fig. 3). Aldolase A is the predominant activity detected in the early rat embryo; A to B and A to C transitions have previously been observed to occur with rat liver and brain embryogenesis, respectively (Weber, 1965; Weber and Rutter, 1964; Rensing et al., 1967). In contrast to the above observations, Masters (1968) has reported appreciable levels of aldolase C in the early embryos of four mammalian species, including rat. The discrepancy between the observations from these different laboratories has yet to be reconciled. Aldolase C is the predominant activity of the early chick embryo and, with development, C to A (Lebherz and Rutter, 1969; Herskovitz et al., 1967) and C to B (present studies) transitions occur in muscle (also heart) and liver, respectively (Fig. 3). The apparent interchangeability of aldolases A and C in the early vertebrate embryo as CHICK
RAT
A
c CA
Al
<
8
--=--,
FIG. 3. Aldolase transitions associated with rat and chick tissue embryogenesis and tissue disease states. See text for details on transitions.
VOLUME 27. 1972
well as in some adult tissues (Lebherz and Rutter, 1969) may suggest a functional equivalence of these two enzymes. Apparent reversals of the aldolase transitions associated with embryogenesis have been observed to be associated with certain disease states (Fig. 3). The B to A transitions associated with invasive rat liver hepatomas have been extensively studied (Rutter et al., 1963a; Matsushima et al., 1968; Ikehara et al., 1970; Gracy et al., 1970). Sugimura and associates (1969) have observed a C to A transition in human brain meningioma. Finally, Schapira et al. (1968) have reported that an A to C transition is associated with muscular dystrophy in the chick. In view of these observations, coupled with the present studies, it is predicted that a reversal of the C to B transition described here, that is, a B to C transition, will be observed by studying chick liver hepatoma tissues. Research supported by the Swiss National Science Foundation, project No. 3.247.69. I would like to thank Drs. H. Ursprung, H. Eppenberger, and D. Turner for helpful discussions during this investigation and Mr. C. Holderegger for his assistance with photography. REFERENCES BALLARD, F. J., and OLIVER, I. T. (1963). Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. Biophys. Acta 71, 578-588. BENOY, M. P., and ELLIOTT, K. A. C. (1937). The metabolism of lactic and pyruvic acids in normal and tumour tissues. V. Synthesis of carbohydrates. Biochem. J. 31, 1268-12’75. BLOSTEIN, R., and RUTTER, W. J. (1963). Comparative studies of liver and muscle aldolase. II. Immunochemical and chromatographic differences. J. Sol. Chem. 238, 3280-3285. CKOISILLE, Y., and LE DOUARIN, N. M. (1965). Development and regeneration of the liver. In “Organogenesis” (R. DeHaan and H. Ursprung, eds.), pp. 421-466. Halt, Rinehart, and Winston, New York. GRACY, R. W., LACKO, A. G., BROX, L. W., ADELMAN, R. C., and HORECKER, B. L. (1970). Structural relations in aldolases purified from rat liver and muscle and Novikoff hepatoma. Arch. Biochem. Biophys. 136, 480-490.
LEBHERZ
Aldolase
HERSKOV~~Z, J. J., MASTERS, C. J., WASSERMAN, P. M., and KAPLAN, N. 0. (1967). On the tissue specificity and biological significance of aldolase C in the chick. Biochem. Biophys. Res. Commurz. 26, 24-29. IKEHARA, Y., ENDO, H., and OKADA, Y. (1970). The identity of the aldolases isolated from rat muscle and primary hepatoma. Arch. Biochem. Biophys. 136, 491-497. KAWAHARA, K., and TANFORD, C. (1966). The number of polypeptide chains in rabbit muscle aldolase. Biochemistry 5, 15781584. KREBS, H. A., BENNETT, D. A. H., DE GASQUET, P., GASCOYNE, T., and YOS~IDA, T. (1963). Renal gluconeogenesis. The effect of diet on the gluconeogenic capacity of rat-kidney-cortex slices. Biochem. J. 86, 22-27. LEBHERZ, H. G., and RUTTER, W. J. (1969). Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 8.109-121. MARQUARDT, R. R. (1969). Multiple molecular forms of avian aldolases. II. Enzymatic properties and amino acid composition of chicken (Gallus domesticus) breast muscle aldolase. Can. J. Biochem. 47, 527-534. MARQUARDT, R. R. (1970). Multiple molecular forms of avian aldolases. IV. Purification and properties of chicken (Gallus domesticus) brain aldolase. Can. J. Biochem. 48, 322-333. MARQUARDT, R. R. (1971a). Multiple molecular forms of avian aldolases. V. Purification and molecular properties of chicken (G&us domesticus) liver aldolase. Can J. Biochem. 49, 647-657. MARQUARDT, R. R. (1971b). Multiple molecular forms of avian aldolases. VI. Enzymatic properties and amino acid composition of chicken liver aldolase and comparative immunochemical properties. Can. J. Biochem. 49, 658-665. MASTERS, C. J. (1968). The ontogeny of mammalian fructose-1,6-diphosphate aldolase. Biochim. Biophys. Acta 167, 161-171. MATSUSHIMA, T., KAWABE, S., SKIBUYA, M., and SUGIMURA, T. (1968). Aldolase isozymes in rat tumour cells. Biochem. Biophys. Res. Commun. 30,565-570. NORDMANN, Y., SCHAPIRA, F., and DREYFUS, J. (1968). A structural modified liver aldolase in fructose intolerance. Immunological and kinetic evidence. Biochem. Biophys. Res. Commun. 31, 884-889. PENHOET, E., RAJKUMAR, T., and RUT~ER, W. J. (1966). Multiple forms of fructose diphosphate aldolase in mammalian tissues. Proc. Nat. Acad. Sci. U. S. 56, 1275-1282. PENHOET. E. E.. and RUITER. W. J. (1971). Catalvtic and immunochemical properties of homomeric and heteromeric combinations of aldolase subunits. J.
B in Chick Embryo
149
Biol. Chem. 246, 318-323. PENHOET, E., KOCHMAN, M., VALENTINE, R., and RU?TER, W. J. (1967). The subunit structure of mammalian fructose diphosphate aldolase. Biochemistrt)? 6, 2940-2949. PENHOET, E. E., KOCHMAN, M., and RUTTER, W. J. (1969a). Isolation of fructose diphosphate aldolases A, B, and C. Biochemistry 8, 4391-4395. PENHOET, E. E., KOCHMAN, M., and RUWER, W. J. (1969b). Molecular and catalytic properties of aldolase C. Biochemistry 8, 4396-4402. RAJKUMAR, T. V., WOODFIN, B. M., and RUTTER, W. J. (1967). Aldolase B from (adult) rabbit liver. Methods Enzymol. 9, 491-498. RENSING, U., SCHMID, A., and LEUTHARDT, F. (1967). Veranderungen im Isoenzymmuster der Aldolasen aus Ratten-Organen wlhrend der Entwicklung. Hoppe-Seyler’s Z. Physiol. Chem. 348, 921-928. RUITER, W. J., and WEBER, C. S. (1965) Specific proteins in cytodifferentiation. In “Developmental and Metabolic Control Mechanisms in Neoplasia” (D. N. Ward, ed.), pp. 195-218. Williams and Wilkins, Baltimore, Maryland. RUT~ER, W. J., BLOSTEIN, R. E., WOODFIN, B. M., and WEBER, C. S. (1963a). Enzyme variants and metabolic diversification. Aduan. Enzyme Regul. 1, 39-56. RULER, W. J., WOODFIN, B. M., and BLOSTEIN, R. E. (1963b). Enzymic homology; structure and catalytic differentiation of fructose diphosphate aldolase. Acta Chem. Stand. 17, 8226-8232. RU’ITER, W. J. RAJKUMAR, T., PENHOET, E., KOCHMAN, M., and VALENTINE, R. (1968). Aldolase variants: Structure and physiological significance. Ann. N. Y. Acad. Sci. 151, 1022117. SCHAPIRA, F., DREYFUS, J., ALLARD, D., and GREGORI-LAUER, C. (1968). Isoenzymes of creatine kinase and aldolase in fetal and pathological muscle. Cle. Chim. Acta 20, 439-447. SUGIMURA, T., SATO, S., KAWABE, S., SUZUKI, N., CHIEN, T. C., and TAMKURA, K. (1969). Aldolase C in brain tumour. Nature (London) 222, 1070. TAYLOR, J. F., GREEN, A. A., and CORI, G. T. (1948). Crystalline aldolase. J. Biol. Chem. 173, 591-604. WEBER, C. S. (1965). Fructose diphosphate aldolase homologs in embryological development. Ph.D. thesis, University of Illinois, Urbana, Illinois. WEBER, C. S., and RUTTER, W. J. (1964). Differential synthesis of aldolases A and B during embryological development. Fed. PFOC. Fed. Amer. Sot. Enp. Biol. 23, 487. WILLIER, B. H. (1968). Glycogen synthesis, storage and transport mechanisms in the yolk-sac membrane of the chick embryo. Wilhelm Roux’ Arch. Entwicklurzgsmech. Organismen 161, 89-117.
BIOLOGY
The Ontogeny
27, 143-149 (1%‘)
of Fructose
Diphosphate
Aldolase
I3 in the
Chick
HERBERT G. LEBHERZ Laboratory
for Developmental
Biology,
Zoology Institute, Zurich, Switzerland
Accepted
Swiss Fedeml Institute
of Technology,
September 27, 1971
The three homologous parental fructose-l, 6-diphosphate aldolases A, B, and C have previously been demonstrated in vertebrates. Although five-membered A-B, A-C, and B-C hybrid sets are readily produced in vitro by reversible dissociation of the appropriate parental enzymes, only A-B and A-C sets have been found heretofore in vertebrate tissues. In the present studies, cellulose-polyacetate electrophoresis followed by specific enzymatic staining has demonstrated the existence of B-C hybrids in uiuo. B-C hybrids were found in early embryonic chick liver (51 to 8h days) and in yolk sac membranes. The aldolase activity profiles suggest a transition in synthesis from aldolase C to predominantlyaldolise B subunits during early chick liver embryogenesis. The fact that B-C hybrids were detected suggests that the C to B transition takes place within individual cells and is not due to the preferential proliferation of cells containing aldolase B. The timing of the C to B transition in liver is consistent with the proposed role of aldolase B in gluconeogenesis. Finally, the present studies are discussed in relation to other aldolase transitions observed to be associated with vertebrate tissue embryogenesis and with certain disease states.
Subunits of aldolases A, B, and C can interact to form hybrid molecules. Since these aldolases are composed of four subunits (Kawahara and Tanford, 1966; Penhoet et al., 1967), five-membered hybrid sets are formed by the binary combination of the parental subunits into tetramers. Five-membered hybrid sets are readily produced in vitro by reversible acid dissociation of the appropriate parental enzymes (Penhoet et al., 1966) and are observed in tissues in which two of the parental enzymes are synthesized simultaneously (Lebherz and Rutter, 1969). Although A-B, A-C, and B-C hybrid sets are readily produced in vitro (Penhoet et al., 1966) only A-B and A-C sets have been observed in vivo. B-C hybrids have never been detected in animal tissues even though certain organs (i.e., chicken kidney) contain both the A-B and A-C sets (Lebherz and Rutter, 1969). The distinctive catalytic properties of aldolases A and B suggest that, in vivo, these enzymes have different physiological functions. Aldolase A appears to be tailored for a glycolytic role while aldolase B
INTRODUCTION
The three parental fructose-l, 6-diphosphate (FDP) aldolases were first isolated from mammalian tissues: aldolase A, the classical muscle enzyme (Taylor et al., 1948); aldolase B, from liver (Rajkumar et al., 1967); and, aldolase C, from brain (Penhoet et al., 1966, 1969a). Although these enzymes are similar in many respects, they can be distinguished on the basis of their catalytic, molecular, electrophoretic, and immunological properties (Rutter et al., 1963b, 1968; Penhoet et al., 1969b). Homologs of the rabbit A, B, and C enzymes have more recently been isolated from chicken tissues and appear to be quite similar to the corresponding rabbit aldolases (Marquardt, 1969, 1970; 1971a, b). In addition, extensive phylogenetic studies have demonstrated the presence of the three parental enzymes, and hence their structural genes, in a wide variety of vertebrates (Lebherz and Rutter, 1969). It seems likely, therefore, that aldolases A, B, and C are present in all vertebrate organisms. 143 Copyright
0 1972 by Academic
Press, Inc.
144
DEVELOPMENTAL BIOLOGY
may function mainly in gluconeogenesis and fructuse metabolism (Rutter et al., 1963a). Both liver and kidney cortex are rich in gluconeogenic activity and the metabolism of fructose via fructose l-phosphate (F-l-P) occurs primarily in liver (Benoy and Elliott, 1937; Krebs et al., 1963). The observed restricted tissue distribution of aldolase B to vertebrate liver and kidney (Lebherz and Rutter, 1969) and the observed lack of functional aldolase B in patients suffering from fructose intolerance disease (Nordmann et al., 1968) are in accordance with the proposed physiological functions of aldolase B. There is yet no known catalytic basis to suggest that aldolases A and C have distinct physiological functions within the cell (Penhoet et al., 196913). The observations that the early chick embryo contains predominantly aldolase C while aldolase B is the predominant form found in adult liver (Lebherz and Rutter, 1969) suggest that a transition in synthesis from aldolase C to B subunits and, hence, the generation of B-C hybrids may occur during chick liver embryogenesis. The present studies demonstrate the presence of B-C hybrid aldolases in early embryonic chick liver (5-8 days) and in yolk sac membranes. In addition, the data suggest that a transition in synthesis from aldolase C to predominantly aldolase B subunits takes place during early chick liver embryogenesis. Finally, the timing of the C to B transition is consistent with the proposed role of aldolase B in gluconeogenesis. MATERIALS
AND
METHODS
Purified enzymes, coenzymes, substrates, and histochemical reagents were obtained from either the Sigma Chemical Co., St. Louis, Missouri, or Calbiochem Inc., Los Angeles, California. Materials for electrophoresis including Sepraphore III cellulose-polyacetate strips were purchased from the Gelman Instrument Co.,
VOLUME 27, 1972
Ann Arbor, Michigan. All other chemicals were of reagent grade. Embryonic chick tissues were obtained from eggs incubated at 38.2”C for appropriate times. Tissues were homogenized at 4°C in approximately 2 volumes of 0.06 M sodium barbital, 0.06% 2-mercaptoethanol, pH 8.6, either in depression wells with a glass rod or in a glass-glass hand homogenizer. Because of their small volumes, homogenates of early embryonic tissues were generally used without further treatment, but care was taken to omit particulate matter from the samples. All yolk sac membrane homogenates were centrifuged at 17,500 g (2-4°C) for 50 min and the supernatants were used for study. Determination of aldolase activity using either 2.5 mM FDP or 10 mM F-l-P as substrate was made by the spectrophotometric method of Blostein and Rutter (1963) in 0.06 M Tris HCl, pH 7.6 at 25°C. A unit of activity is expressed as the cleavage of 1 pmole of substrate per minute per milliter of extract. Cellulose-polyacetate electrophoresis was performed in 0.06 M sodium barbital, 0.06% 2-mercaptoethanol, pH 8.6 according to the method of Penhoet et al. (1966). Tissue homogenate, 2-5 ~1 containing 0.001 to 0.005 unit of aldolase activity, was applied to the strips and electrophoresis was performed at 250 V for 90 min. The strips were then stained for FDP cleavage activity as described by Penhoet et al. (1966). Location of F-l-P cleavage activity was determined by incubating strips with staining gels containing 0.02 M F-l-P and 0.1 mg/ml ol-glycerolphosphate dehydrogenase-triose phosphate isomerase in place of the 0.01 M FDP used above. The strips and staining gels were incubated at 37°C until good color development (Formazan production) occurred. RESULTS
The parental aldolases A, B, and C from those from most vertebrates, including
LEBHERZ
Aldolase
chicken, have different electrophoretic mobilities (Lebherz and Rutter, 1969). In the present studies, the parental and hybrid aldolases were resolved by cellulosepolyacetate electrophoresis and located by specific enzymatic staining. Positive identification of the activities was made on the basis of electrophoretic mobility and substrate specificity. Substrate specificity is particularly useful in distinguishing aldolase B activity from aldolase A and C activity. Aldolase B has a considerably lower K, for fructose l-phosphate (F-l-P) than do aldolases A and C. Furthermore, aldolase B functions with the same efficiency with either FDP or F-l-P as substrate (FDP/F-1-P = 1). In contrast, aldolases A and C function more efficiently with FDP and have FDP/F-1-P activity ratios of 10 or greater (Rutter et al., 1968; Penhoet et al., 1969b). The relatively high efficiency of F-l-P cleavage exhibited by aldolase B is presumably a reflection of this enzyme’s function in the metabolism of fructose via fructose lphosphate (Rutter et al., 1963a) and, therefore, is a suitable diagnostic parameter for aldolase B activity. Since the catalytic properties of aldolase subunits are apparently the same whether they exist in homomeric or heteromeric (hybrid) combinations (Penhoet and Rutter, 1971), the relative intensities of aldolase activities stained with FDP and F-l-P can be used to positively identify those tetramers which contain B subunits. Figure 1 demonstrates the resolution and identification of aldolase activities in 17-day-chick liver and skeletal muscle. When the strips are stained with FDP (top) the entire A-C hybrid set (A,, A,C, A2C,, AC,, C,) is readily apparent in the muscle profile while aldolase B, which migrates more cathodally than the other activities, is the predominant enzyme found in liver. In contrast, when similar strips are stained with F-l-P, only aldolase B of liver is developed (bottom). The FDP and F-l-P activity profiles of
B in Chick Embryo
145
FIG. 1. Aldolase activity profiles of 17.day chick leg skeletal muscle (M) and liver (L) using fructose1,6-disphosphate (FDP) (top) and fructose l-phosphate (F-1-P) (bottom) as substrates. See Materials and Methods section for details. Muscle and liver extracts were electrophoresed on the same strips.
liver and heart from early chick embryos are presented in Fig. 2. The 17-day muscle profiles are included for comparison. All heart samples contained the A-C activities Cd, ACB, and A&,. These same activities were observed in total homogenates of 3-day embryos. The transition from predominantly aldolase C to the entire A-C set which is present in adult heart (Lebherz and Rutter, 1969) takes place after 17 days of incubation, and probably occurs mainly after hatching. Notice that the activities of muscle and heart are not revealed with F-l-P. The two most anodal activities detected in liver homogenates correspond to the enzymes C, and AC, (Fig. 2). The profiles of 4-day liver are essentially identical to those of heart and no aldolase B activity was detected in 4-day liver by staining with F-l-P. In contrast, major activities which do not correspond to members of the A-C set were detected in 5day liver. The three prominent cathodal activities have mobilities expected for the B-C hybrids BC,, B,C,, and B,C. Low levels of the homotetramer B,, were also detected with FDP. Positive identification of these activities as members of the B-C set was made from the F-l-P profiles. As shown, activities corresponding to B,, B,C, B2C2, and BC, were readily apparent.
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FDP Q +
FIG. 2. Aldolase activity profiles of embryonic chick tissues (M = muscle, H = heart, I, = liver). All heart and liver extracts of each age were electrophoresed on the same strips. The slight staining at the origin of the 7 day fructose l-phosphate (F-1-P) profile is presumably due to enzyme activity associated with particulate matter. See Materials and Methods section for details.
The 6-day liver contains predominantly aldolases B,, B,C, and B,C,, although B,C, was only weakly detected in the F-l-P profile. The transition toward aldolase B continues during day 7 and is essentially completed by day 8 as suggested by the F-l-P profiles. Aldolase B, was the only major activity detected in the 17-day liver. Very few activities corresponding to A-B or A-B-C hybrids were detected
during the C to B transition. On occasion, the C to B transition was observed to be completed slightly later than the sample shown in Fig. 2. However, in all cases the transition was essentially completed by day 10, as evidenced by activity profiles and the similarity between the FDP/F-lP activity rations of IO-day and adult liver samples (FDP/F-1-P = 1.2-1.3). It is essential to point out that the FDP
LEBHERZ
Aldolase
and F-l-P activity profiles presented above reflect to a great extent the catalytic behavior of the three aldolase subunits. The increased F-l-P staining of the B-C hybrids toward the B terminus is inof fluenced by the “specific activities” B subunits in the various tetramers. For example, the hybrid B,C, exhibits one half of the B activity per tetramer as does the enzyme B,. Conversely, the relative intensities of the activities revealed with FDP deceptively emphasize the molar concentrations of tetramers containing A and C subunits since aldolases A and C have considerably higher FDP turnover numbers than does aldolase B (Penhoet et al., 1969b). Since many of the biochemical activities found in liver are also present in yolk sac membrane (YSM) and since the YSM presumably functions in a liver capacity during early chick development (Croisille and Le Douarin, 1965), it was of interest to determine which aldolases are present in this membrane during early development. Studies similar to those performed on chick liver revealed the presence of aldolase B and B-C hybrids in the YSM of all embryos tested (3-10 days). No aldolase B activity was detected in another membrane, the allantois, embryonic which instead contained the A-C activities Cd, ACB, and A&,. DISCUSSION
The present studies demonstrate for the first time the existence of B-C hybrid aldolases in vivo. These hybrids were detected in early embryonic chick liver (5-8 days) and in yolk sac membranes. The fact that B-C hybrids exist in these tissues suggests that subunits of aldolases B and C are simultaneously synthesized within the same cells. In contrast, the observed lack of B-C hybrids in adult vertebrate organs (Lebherz and Rutter, 1969) indicates that, in differentiated tissues, aldolase B and C subunits are rarely, if ever, synthesized within the same cells at the same time.
B in Chick Embryo
147
Whether or not there is any physiological or other basis for the apparent lack of B-C hybrids in differentiated vertebrate organs is not known. The low levels of A-C activities detected in embryonic and adult liver are presumably located in erythroid or other cells (Rutter et al., 1963a; Rutter and Weber, 1965). The transition from aldolase C to predominatly aldolase B observed between approximately 5 and 8 days of incubation as well as the observed decrease in FDP/F-1-P activity ratio from 17 to 2 during this time (Weber, 1965) presumably reflect a transition in synthesis from predominantly aldolase C to predominantly aldolase B subunits by the early embryonic chick liver. This interpretation is supported by the studies of Weber (1965) and Weber and Rutter (1964) which demonstrated a preferential synthesis of aldolase B subunits during the A to B transition associated with rat liver embryogenesis. other interpretations of the However, one based on present data, including preferential degradation of C over B subunits, cannot be eliminated at this time. The fact that B-C hybrids exist supports the contention that the C to B transition observed occurs within individual cells, presumably parenchymal, and is not due to the preferential proliferation of cells containing aldolase B. The finding of appreciable aldolase B activity in yolk sac membrane is also of interest from a physiological point of view. The YSM and liver possess many biochemical activities in common; moreover, this embryonic membrane appears to carry out liver functions prior to the time liver has the capacity to do so (Willier, 1968; Croisille and Le Douarin, 1965). It is perhaps significant, therefore, that aldolase B activity has been detected in YSM several days prior to the time this enzyme appears in liver. The timing of the C to B transition in chick liver is consistent with the gluconeogenie function assigned to aldolase B. The
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DEVELOPMENTAL BIOLOGY
transition toward aldolase B is completed just prior to the time of marked gluconeogenie activity exhibited by embryonic chick liver between approximately 11 and 15 days of incubation (Ballard and Oliver, 1963). In addition, the A to B transition observed during rat liver embryogenesis, 14 days to birth (Rutter and Weber, 1965; Weber, 1965), is completed just prior to the onset of gluconeogenesis in rat liver observed to occur at birth (Ballard and Oliver, 1963). Therefore, in both organisms high levels of aldolase B are present in liver at the time when appreciable gluconeogenie activity becomes apparent. The present demonstration of a C to B transition during early chick liver embryogenesis supplements earlier studies on the aldolases of developing vertebrate tissues (Fig. 3). Aldolase A is the predominant activity detected in the early rat embryo; A to B and A to C transitions have previously been observed to occur with rat liver and brain embryogenesis, respectively (Weber, 1965; Weber and Rutter, 1964; Rensing et al., 1967). In contrast to the above observations, Masters (1968) has reported appreciable levels of aldolase C in the early embryos of four mammalian species, including rat. The discrepancy between the observations from these different laboratories has yet to be reconciled. Aldolase C is the predominant activity of the early chick embryo and, with development, C to A (Lebherz and Rutter, 1969; Herskovitz et al., 1967) and C to B (present studies) transitions occur in muscle (also heart) and liver, respectively (Fig. 3). The apparent interchangeability of aldolases A and C in the early vertebrate embryo as CHICK
RAT
A
c CA
Al
<
8
--=--,
FIG. 3. Aldolase transitions associated with rat and chick tissue embryogenesis and tissue disease states. See text for details on transitions.
VOLUME 27. 1972
well as in some adult tissues (Lebherz and Rutter, 1969) may suggest a functional equivalence of these two enzymes. Apparent reversals of the aldolase transitions associated with embryogenesis have been observed to be associated with certain disease states (Fig. 3). The B to A transitions associated with invasive rat liver hepatomas have been extensively studied (Rutter et al., 1963a; Matsushima et al., 1968; Ikehara et al., 1970; Gracy et al., 1970). Sugimura and associates (1969) have observed a C to A transition in human brain meningioma. Finally, Schapira et al. (1968) have reported that an A to C transition is associated with muscular dystrophy in the chick. In view of these observations, coupled with the present studies, it is predicted that a reversal of the C to B transition described here, that is, a B to C transition, will be observed by studying chick liver hepatoma tissues. Research supported by the Swiss National Science Foundation, project No. 3.247.69. I would like to thank Drs. H. Ursprung, H. Eppenberger, and D. Turner for helpful discussions during this investigation and Mr. C. Holderegger for his assistance with photography. REFERENCES BALLARD, F. J., and OLIVER, I. T. (1963). Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. Biophys. Acta 71, 578-588. BENOY, M. P., and ELLIOTT, K. A. C. (1937). The metabolism of lactic and pyruvic acids in normal and tumour tissues. V. Synthesis of carbohydrates. Biochem. J. 31, 1268-12’75. BLOSTEIN, R., and RUTTER, W. J. (1963). Comparative studies of liver and muscle aldolase. II. Immunochemical and chromatographic differences. J. Sol. Chem. 238, 3280-3285. CKOISILLE, Y., and LE DOUARIN, N. M. (1965). Development and regeneration of the liver. In “Organogenesis” (R. DeHaan and H. Ursprung, eds.), pp. 421-466. Halt, Rinehart, and Winston, New York. GRACY, R. W., LACKO, A. G., BROX, L. W., ADELMAN, R. C., and HORECKER, B. L. (1970). Structural relations in aldolases purified from rat liver and muscle and Novikoff hepatoma. Arch. Biochem. Biophys. 136, 480-490.
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HERSKOV~~Z, J. J., MASTERS, C. J., WASSERMAN, P. M., and KAPLAN, N. 0. (1967). On the tissue specificity and biological significance of aldolase C in the chick. Biochem. Biophys. Res. Commurz. 26, 24-29. IKEHARA, Y., ENDO, H., and OKADA, Y. (1970). The identity of the aldolases isolated from rat muscle and primary hepatoma. Arch. Biochem. Biophys. 136, 491-497. KAWAHARA, K., and TANFORD, C. (1966). The number of polypeptide chains in rabbit muscle aldolase. Biochemistry 5, 15781584. KREBS, H. A., BENNETT, D. A. H., DE GASQUET, P., GASCOYNE, T., and YOS~IDA, T. (1963). Renal gluconeogenesis. The effect of diet on the gluconeogenic capacity of rat-kidney-cortex slices. Biochem. J. 86, 22-27. LEBHERZ, H. G., and RUTTER, W. J. (1969). Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 8.109-121. MARQUARDT, R. R. (1969). Multiple molecular forms of avian aldolases. II. Enzymatic properties and amino acid composition of chicken (Gallus domesticus) breast muscle aldolase. Can. J. Biochem. 47, 527-534. MARQUARDT, R. R. (1970). Multiple molecular forms of avian aldolases. IV. Purification and properties of chicken (Gallus domesticus) brain aldolase. Can. J. Biochem. 48, 322-333. MARQUARDT, R. R. (1971a). Multiple molecular forms of avian aldolases. V. Purification and molecular properties of chicken (G&us domesticus) liver aldolase. Can J. Biochem. 49, 647-657. MARQUARDT, R. R. (1971b). Multiple molecular forms of avian aldolases. VI. Enzymatic properties and amino acid composition of chicken liver aldolase and comparative immunochemical properties. Can. J. Biochem. 49, 658-665. MASTERS, C. J. (1968). The ontogeny of mammalian fructose-1,6-diphosphate aldolase. Biochim. Biophys. Acta 167, 161-171. MATSUSHIMA, T., KAWABE, S., SKIBUYA, M., and SUGIMURA, T. (1968). Aldolase isozymes in rat tumour cells. Biochem. Biophys. Res. Commun. 30,565-570. NORDMANN, Y., SCHAPIRA, F., and DREYFUS, J. (1968). A structural modified liver aldolase in fructose intolerance. Immunological and kinetic evidence. Biochem. Biophys. Res. Commun. 31, 884-889. PENHOET, E., RAJKUMAR, T., and RUT~ER, W. J. (1966). Multiple forms of fructose diphosphate aldolase in mammalian tissues. Proc. Nat. Acad. Sci. U. S. 56, 1275-1282. PENHOET. E. E.. and RUITER. W. J. (1971). Catalvtic and immunochemical properties of homomeric and heteromeric combinations of aldolase subunits. J.
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Biol. Chem. 246, 318-323. PENHOET, E., KOCHMAN, M., VALENTINE, R., and RU?TER, W. J. (1967). The subunit structure of mammalian fructose diphosphate aldolase. Biochemistrt)? 6, 2940-2949. PENHOET, E. E., KOCHMAN, M., and RUTTER, W. J. (1969a). Isolation of fructose diphosphate aldolases A, B, and C. Biochemistry 8, 4391-4395. PENHOET, E. E., KOCHMAN, M., and RUWER, W. J. (1969b). Molecular and catalytic properties of aldolase C. Biochemistry 8, 4396-4402. RAJKUMAR, T. V., WOODFIN, B. M., and RUTTER, W. J. (1967). Aldolase B from (adult) rabbit liver. Methods Enzymol. 9, 491-498. RENSING, U., SCHMID, A., and LEUTHARDT, F. (1967). Veranderungen im Isoenzymmuster der Aldolasen aus Ratten-Organen wlhrend der Entwicklung. Hoppe-Seyler’s Z. Physiol. Chem. 348, 921-928. RUITER, W. J., and WEBER, C. S. (1965) Specific proteins in cytodifferentiation. In “Developmental and Metabolic Control Mechanisms in Neoplasia” (D. N. Ward, ed.), pp. 195-218. Williams and Wilkins, Baltimore, Maryland. RUT~ER, W. J., BLOSTEIN, R. E., WOODFIN, B. M., and WEBER, C. S. (1963a). Enzyme variants and metabolic diversification. Aduan. Enzyme Regul. 1, 39-56. RULER, W. J., WOODFIN, B. M., and BLOSTEIN, R. E. (1963b). Enzymic homology; structure and catalytic differentiation of fructose diphosphate aldolase. Acta Chem. Stand. 17, 8226-8232. RU’ITER, W. J. RAJKUMAR, T., PENHOET, E., KOCHMAN, M., and VALENTINE, R. (1968). Aldolase variants: Structure and physiological significance. Ann. N. Y. Acad. Sci. 151, 1022117. SCHAPIRA, F., DREYFUS, J., ALLARD, D., and GREGORI-LAUER, C. (1968). Isoenzymes of creatine kinase and aldolase in fetal and pathological muscle. Cle. Chim. Acta 20, 439-447. SUGIMURA, T., SATO, S., KAWABE, S., SUZUKI, N., CHIEN, T. C., and TAMKURA, K. (1969). Aldolase C in brain tumour. Nature (London) 222, 1070. TAYLOR, J. F., GREEN, A. A., and CORI, G. T. (1948). Crystalline aldolase. J. Biol. Chem. 173, 591-604. WEBER, C. S. (1965). Fructose diphosphate aldolase homologs in embryological development. Ph.D. thesis, University of Illinois, Urbana, Illinois. WEBER, C. S., and RUTTER, W. J. (1964). Differential synthesis of aldolases A and B during embryological development. Fed. PFOC. Fed. Amer. Sot. Enp. Biol. 23, 487. WILLIER, B. H. (1968). Glycogen synthesis, storage and transport mechanisms in the yolk-sac membrane of the chick embryo. Wilhelm Roux’ Arch. Entwicklurzgsmech. Organismen 161, 89-117.