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Ann. H u m . Genet., L o d . (1976), 39,277
Printed in Great Britain
Enolase : human tissue distribution and evidence for three different loci* BY SHI-HAN CHENi AND ELOISE R. GIBLETTS Enolase (phosphopyruvate hydratase, EC. 4 . 2 . 1 .11) is a hydro-lyase which catalyses the reversible conversion of 2-phosphoglycerate (2 PGA) to phospho-enol pyruvate in the glycolytic pathway. The enzyme is ubiquitous among animal tissues and is believed t o be a dimer with a molecular weight of about 90,000 daltons (Wold, 1971 ; Westhead, 1967). A genetic variant of erythrocyte enolase (ENO) has been recently described (Giblett et al. 1974). Family studies of E N 0 phenotypes suggested co-dominant inheritance, with the structural gene locus being possibly linked to the R h locus on chromosome 1 (Giblett et al. 1974; Lewis & Giblett, 1975, personal communication). Assignment of the E N 0 locus t o chromosome 1 has been independently demonstrated by cell hybridization studies (Meera Khan et al. 1974). I n this communication, we describe E N 0 patterns in several human tissues and present evidence for the existence of two additional E N 0 loci. MATERIALS AND METHODS
Blood specimens were drawn into ACD solution, and saline-washed red cells were stored in glycerol until used. Haemolysates were prepared as previously described (Chen & Giblett, 1971). Specimens of human tissue were obtained a t autopsy including liver, muscle, heart, kidney, brain, intestine, spleen and placenta. Each tissue was prepared for electrophoresis by homogenization of minced tissue in 5 volumes (wlv) of distilled H,O and centrifuged a t 15,900 g for 20 min. a t 4" C. White cell and cultured skin fibroblast extracts were prepared as previously described (Chen & Giblett, 1971 ; Chen, Scott & Swedberg, 1974). Vertical starch gel electrophoresis was performed a t 4" C. for 18 hr. using 0.1 M phosphate buffer, pH 6.5. For cellogel electrophoresis, 0.01 M phosphate buffer, p H 6.2 was used. The staining solution for E N 0 activity contained 0.1 M Tris-HC1 buffer, p H 7.8, 8 mM-MgSO,; 0.1 M-KCl, 1 mM 2 PGA, 2 mM ADP, 1 mM NADH, 10 units per ml. of pyruvate kinase (Sigma Type 11)and 30 units per ml. of lactate dehydrogenase (Sigma Type 11).The sites of EN0 activity appeared as dark bands against a fluorescent background when viewed under longwave ultraviolet light. Enolase activity was assayed by the spectrophotometric procedure described by Holt & Wold (1961) using a Gilford 2400 S spectrophotometer a t 240 nm. A unit of E N 0 activity was defined as the amount converting 1 pmole of substrate per minute. Brain E N 0 was partially purified by the method of Wood (1964). Final separation of the enzyme into 3 components was achieved by chromatographing the protein in a DEAE-Cellulose column (20 x 1.4 cm.) equilibrated with 0-005 M sodium phosphate buffer, p H 6.5, and eluting with a linear saline gradient from 0 to 0.5 RZ NaCl in the equilibrating buffer. Dissociation and reassociation of the purified components were performed in the presence of 1 M NaCl, 0.4 M sucrose, 0.1 M 2-mercaptoethanol, and 0.1 mM
* This work was supported by PHS grants GM-15253, HD-04665, AM-09745, HL-17265 and a grant from the National Foundation. t Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington 98195. $ Puget Sound Blood Center, Terry and Madison, Seattle, Washington 98104.
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Fig. 1
Fig. 2
Fig. 3
Fig. 1. Cellogel electrophoresis showing the electrophoretic patterns of human brain, liver, muscle and red cells, respectively, from left to right. I n channel 4, haemoglobin migrates cathodal to the E N 0 I band. Fig. 2. Photograph and diagram showing electrophoretic patterns of EN0 in 2 control haemolysates (channels 1 and 3) and a variant with EN0 2-1 phenotype (channel 2). The proposed subunit structure of the individual isozymes is indicated on the left. Because of prolonged exposure of the anodal portion to staining solution, band I1 in the control patterns appears to stain as intensely as band I. With equal exposure time, band I is much more intense than band 11, as shown in Fig. 1. Fig. 3. Cellogel eleotrophoretic p a t t e r n of the partially purified EN0 before and after dissociation and reassociation of E N 0 bands I and 111. The pattern in channels 1 and 4 represent purified I and 111, respectively, and that of channel 2 represents a simple mixture of both components. The pattern in channel 3 was obtained after dissociation and rertssociation, as described in the Methods section.
EDTA in 0.1 M phosphate buffer, pH 7.0. Mixtures (about 0.8 unitlml.) of the enzyme components were left overnight at - 20" C., and desalted after thawing by dialysis against 0.01 iv phosphate buffer, pH 6.5. The dialysates were immediately subjected to cellogel electrophoresis followed by staining for EN0 activity. RESULTS
Figure 1 shows the four different EN0 patterns found by cellogel electrophoresis of the tested tissue extracts. I n red cell haemolysates there was one strongly stained band (I)and two minor bands (I1and 111).This pattern was also found in extracts of kidney, white cells and cultured skin fibroblasts. Brain extracts had the same three components as the haemolysates except that bands I1 and I11 were much more heavily stained. A third pattern found in extracts of liver, heart, intestine, spleen and placenta, consisted of a single band with the same mobility as the major red cell band (I).Muscle extracts had the fourth pattern, consisting of a single band (Im) which moved slightly faster than band I found in other tissues. The second channel of Fig. 2 shows the red cell electrophoresisEN0 pattern of a heterozygous individual. I n this EN0 2-1 phenotype, a triple-banded pattern replaces the single band I of the common EN0 1 phenotype, while two zones replace band 11.Band I11is a single weakly stained zone. Figure 3 shows the single I and I11 bands obtained after partial purification and chromatographic separation of a brain extract from an individual of the common EN0 1 phenotype.
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Enolase :human tissue distribution
When these two components were mixed just before electrophoresis, the stained gel had the expected two band pattern (channel 2). However, when the mixture was subjected to dissociation and reassociation, a new isozyme with the intermediate mobility of component I1 was generated (channel 3). No new isozyme was produced when the individual, unmixed components were subjected to the dissociation and reassociation procedure. When purified component I1 was treated in this manner, isozymes with the same mobility as components I and I11were generated.
DISCUSSION
The data presented here strongly support the existence of two independent structural human gene loci for enolase: ENO, and ENO, governing the synthesis of two different polypeptide chains (aand p). These chains form random dimers, so that in individuals with the common EN0 1 phenotype, bands I and I11represent homodimers (aaand pp), while band I1is a heterodimer (ap).This interpretation fits the fact that it was possible to generate a zone corresponding to the heterodimer from the two homodimers, and to produce bands corresponding to the two homodimers from the heterodimer. In individuals with the rare E N 0 2-1 phenotype, the variant ENO; gene produces an a2 peptide which forms a heteromer (a1a2) with the ENO: gene product (a1). The two homodimers (aW and a2az) are also formed, thus producing a three-banded pattern in place of the single band I of the common EN0 1 phenotype. The two zones replacing the single alp dimer of band I1 consist of alp and a2pin the variant phenotype, while the third pp band remains unchanged. On the basis of our study, the slightly faster-moving band of adult muscle extracts could be a secondary modification of band I. However, in the accompanying paper by Pearce, Edwards & Harris, use of a different electrophoretic system clearly distinguishes the muscle form and establishes the existence of a third locus. Rider & Taylor (1974) described one form of enolase in rat muscle, and a second form in liver, brain, blood and other tissues. The study in rats did not detect an anodal brain isozyme analogous to that reported here in human brain. Enolase has been purified from several mammalian tissues, including muscle, brain, liver and erythrocytes (Wood, 1964; Baranowski, Wolna & Morawiecki, 1968; Witt & Witz, 1970; Rider & Taylor, 1974). In the single report describing the enzyme in brain tissue (Wood, 1964), the physical and chemical properties were very similar to those of enolase preparations from other tissues of various mammals. Human brain tissue appears to be the best source for the second locus product (i.e. band 111), although lower concentrations of this ,8p isozyme are present in other tissues, as well as in the cultured skin fibroblasts. It should therefore be possible to assign the second E N 0 locus to its chromosome by somatic cell hybridization (Ruddle, 1972). SUMMARY
Four different cellogel electrophoretic patterns of enolase were found in human tissue extracts. They consisted cf: (A) one strongly stained band (I)and two minor bands (I1and 111)found in haemolysates, white cell, skin fibroblast and kidney extracts ; (B) a three-banded pattern in brain resembling that of haemolysates except for heavier concentrations of bands I1 and 111; 18
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(C) a single component corresponding to band I, found in liver, heart, intestine, spleen and placenta ; (D) a single band with slightly faster mobility than band I, found in adult muscle extracts. The haemolysate of an individual with the heterozygous ENOilEN02, genotype had a triplebanded pattern replacing band I, a double-banded pattern replacing band I1 and a single weakly staining band 111.This finding, as well as the results of dissociation and recombination experiments, supports the hypothesis that the enzyme is a dimer formed by random interaction of two polypeptide chains (aand /3) synthesized by two independent gene loci (ENO, and ENO,). Evidence for a third locus, ENO,, is supplied by the electrophoretic pattern of muscle extracts. We thank Dr C. Ronald Scott, Department of Pediatrics, University of Washington, for supplying us adult post-mortem tissues and for reviewing the manuscript; and Dr Gilbert S. Omenn, Department of Medicine, University of Washington, for supplying some of the tissue extracts in this study. REFERENCES
BARANOWSKI, T., WOLNA, E. & MORAWIECIU, A. (1968). Purification and properties of crystalline a-phosphoD glycerate hydro-lyase from human muscle. Eur. J . Biochem. 5, 119. CIIEN, S.-H. & GIBLETT,E. R. (1971). Genetic variation of soluble glutamic-oxaloacetic transaminase in man. Am. J . Hum. Genet. 23,419. CHEN, 8.-H., SCOTT,C. R. & SWEDBERC, K. R. (1974). Heterogeneity of adenosine deaminase deficiency: expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells. Am. J . Hum. Genet. 27, 46.
GIBLETT, E. R., CHEN, S.-H., ANDERSON, J. E. & LEWIS, M. (1974). Afamily study suggesting genetic linkage of phospho-pyruvate hydratase (Enolase) to the Rh blood group system. Cytogenet. Cell Genet. 13, 91. HOLT, A. & WOLD,F. (1961). The isolation and characterization of rabbit muscle enolase. J . Biol. Chem. 236, 3227.
A. & WOSTERVELD,A. (1974). The human loci for MEERA KHAN,P., DOPPERT,B. A., HAOEMEIZER, phosphopyruvate hydratase and guanylate kinase are syntenic with the PGD-PGM, linkage groups in man-Chinese hamster somatic cell hybrids. Cytogenet. Cell Genet. 13, 130. PEARCE,J. M., EDWARDS, Y. H. & HARRIS,H. (1975). Human enolase isozyme: electrophoresis and biochemical evidence for three loci. Ann. Hum. Genet. 39, 363. RIDER,C. C. & TAYLOR,C. B. (1974). Enolase isozymes in rat tissues: electrophoretic chromatographic, immunological and kinetic properties. Bwchim. Biophys. Acta 365, 285. RUDDLE, F. H. (1972). In Advances i n Human Genetice 3 (ed. H. Harris and K. Hirschhorn). New York: Plenum Press. WESTHEAD, E. D. (1967). I n Methods i n Enzymology Z X (ed. S. P. Colowck), p. 670. New York: Academic Press. WITT, I. & WITZ, D. (1970). Reinigung und Charakterisierung von Phosphopyruvate-hydratase(enolase: E.C.4.2.1.11) aus Neugeborenen- und Erwachsenen-Erythrozyten. Zeit. PhySioZ. Chem. 351, 1232. WOLD,F. (1971). In The Enzymes I.' (ed. P. Boyer), p. 499. New York and London: Academic Press. WOOD, T. (1964). The purification of enolase from cerebral tissue. Biochm. J . 91, 453.
Ann. H u m . Genet., L o d . (1976), 39,277
Printed in Great Britain
Enolase : human tissue distribution and evidence for three different loci* BY SHI-HAN CHENi AND ELOISE R. GIBLETTS Enolase (phosphopyruvate hydratase, EC. 4 . 2 . 1 .11) is a hydro-lyase which catalyses the reversible conversion of 2-phosphoglycerate (2 PGA) to phospho-enol pyruvate in the glycolytic pathway. The enzyme is ubiquitous among animal tissues and is believed t o be a dimer with a molecular weight of about 90,000 daltons (Wold, 1971 ; Westhead, 1967). A genetic variant of erythrocyte enolase (ENO) has been recently described (Giblett et al. 1974). Family studies of E N 0 phenotypes suggested co-dominant inheritance, with the structural gene locus being possibly linked to the R h locus on chromosome 1 (Giblett et al. 1974; Lewis & Giblett, 1975, personal communication). Assignment of the E N 0 locus t o chromosome 1 has been independently demonstrated by cell hybridization studies (Meera Khan et al. 1974). I n this communication, we describe E N 0 patterns in several human tissues and present evidence for the existence of two additional E N 0 loci. MATERIALS AND METHODS
Blood specimens were drawn into ACD solution, and saline-washed red cells were stored in glycerol until used. Haemolysates were prepared as previously described (Chen & Giblett, 1971). Specimens of human tissue were obtained a t autopsy including liver, muscle, heart, kidney, brain, intestine, spleen and placenta. Each tissue was prepared for electrophoresis by homogenization of minced tissue in 5 volumes (wlv) of distilled H,O and centrifuged a t 15,900 g for 20 min. a t 4" C. White cell and cultured skin fibroblast extracts were prepared as previously described (Chen & Giblett, 1971 ; Chen, Scott & Swedberg, 1974). Vertical starch gel electrophoresis was performed a t 4" C. for 18 hr. using 0.1 M phosphate buffer, pH 6.5. For cellogel electrophoresis, 0.01 M phosphate buffer, p H 6.2 was used. The staining solution for E N 0 activity contained 0.1 M Tris-HC1 buffer, p H 7.8, 8 mM-MgSO,; 0.1 M-KCl, 1 mM 2 PGA, 2 mM ADP, 1 mM NADH, 10 units per ml. of pyruvate kinase (Sigma Type 11)and 30 units per ml. of lactate dehydrogenase (Sigma Type 11).The sites of EN0 activity appeared as dark bands against a fluorescent background when viewed under longwave ultraviolet light. Enolase activity was assayed by the spectrophotometric procedure described by Holt & Wold (1961) using a Gilford 2400 S spectrophotometer a t 240 nm. A unit of E N 0 activity was defined as the amount converting 1 pmole of substrate per minute. Brain E N 0 was partially purified by the method of Wood (1964). Final separation of the enzyme into 3 components was achieved by chromatographing the protein in a DEAE-Cellulose column (20 x 1.4 cm.) equilibrated with 0-005 M sodium phosphate buffer, p H 6.5, and eluting with a linear saline gradient from 0 to 0.5 RZ NaCl in the equilibrating buffer. Dissociation and reassociation of the purified components were performed in the presence of 1 M NaCl, 0.4 M sucrose, 0.1 M 2-mercaptoethanol, and 0.1 mM
* This work was supported by PHS grants GM-15253, HD-04665, AM-09745, HL-17265 and a grant from the National Foundation. t Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington 98195. $ Puget Sound Blood Center, Terry and Madison, Seattle, Washington 98104.
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278
+ 111
Fig. 1
Fig. 2
Fig. 3
Fig. 1. Cellogel electrophoresis showing the electrophoretic patterns of human brain, liver, muscle and red cells, respectively, from left to right. I n channel 4, haemoglobin migrates cathodal to the E N 0 I band. Fig. 2. Photograph and diagram showing electrophoretic patterns of EN0 in 2 control haemolysates (channels 1 and 3) and a variant with EN0 2-1 phenotype (channel 2). The proposed subunit structure of the individual isozymes is indicated on the left. Because of prolonged exposure of the anodal portion to staining solution, band I1 in the control patterns appears to stain as intensely as band I. With equal exposure time, band I is much more intense than band 11, as shown in Fig. 1. Fig. 3. Cellogel eleotrophoretic p a t t e r n of the partially purified EN0 before and after dissociation and reassociation of E N 0 bands I and 111. The pattern in channels 1 and 4 represent purified I and 111, respectively, and that of channel 2 represents a simple mixture of both components. The pattern in channel 3 was obtained after dissociation and rertssociation, as described in the Methods section.
EDTA in 0.1 M phosphate buffer, pH 7.0. Mixtures (about 0.8 unitlml.) of the enzyme components were left overnight at - 20" C., and desalted after thawing by dialysis against 0.01 iv phosphate buffer, pH 6.5. The dialysates were immediately subjected to cellogel electrophoresis followed by staining for EN0 activity. RESULTS
Figure 1 shows the four different EN0 patterns found by cellogel electrophoresis of the tested tissue extracts. I n red cell haemolysates there was one strongly stained band (I)and two minor bands (I1and 111).This pattern was also found in extracts of kidney, white cells and cultured skin fibroblasts. Brain extracts had the same three components as the haemolysates except that bands I1 and I11 were much more heavily stained. A third pattern found in extracts of liver, heart, intestine, spleen and placenta, consisted of a single band with the same mobility as the major red cell band (I).Muscle extracts had the fourth pattern, consisting of a single band (Im) which moved slightly faster than band I found in other tissues. The second channel of Fig. 2 shows the red cell electrophoresisEN0 pattern of a heterozygous individual. I n this EN0 2-1 phenotype, a triple-banded pattern replaces the single band I of the common EN0 1 phenotype, while two zones replace band 11.Band I11is a single weakly stained zone. Figure 3 shows the single I and I11 bands obtained after partial purification and chromatographic separation of a brain extract from an individual of the common EN0 1 phenotype.
279
Enolase :human tissue distribution
When these two components were mixed just before electrophoresis, the stained gel had the expected two band pattern (channel 2). However, when the mixture was subjected to dissociation and reassociation, a new isozyme with the intermediate mobility of component I1 was generated (channel 3). No new isozyme was produced when the individual, unmixed components were subjected to the dissociation and reassociation procedure. When purified component I1 was treated in this manner, isozymes with the same mobility as components I and I11were generated.
DISCUSSION
The data presented here strongly support the existence of two independent structural human gene loci for enolase: ENO, and ENO, governing the synthesis of two different polypeptide chains (aand p). These chains form random dimers, so that in individuals with the common EN0 1 phenotype, bands I and I11represent homodimers (aaand pp), while band I1is a heterodimer (ap).This interpretation fits the fact that it was possible to generate a zone corresponding to the heterodimer from the two homodimers, and to produce bands corresponding to the two homodimers from the heterodimer. In individuals with the rare E N 0 2-1 phenotype, the variant ENO; gene produces an a2 peptide which forms a heteromer (a1a2) with the ENO: gene product (a1). The two homodimers (aW and a2az) are also formed, thus producing a three-banded pattern in place of the single band I of the common EN0 1 phenotype. The two zones replacing the single alp dimer of band I1 consist of alp and a2pin the variant phenotype, while the third pp band remains unchanged. On the basis of our study, the slightly faster-moving band of adult muscle extracts could be a secondary modification of band I. However, in the accompanying paper by Pearce, Edwards & Harris, use of a different electrophoretic system clearly distinguishes the muscle form and establishes the existence of a third locus. Rider & Taylor (1974) described one form of enolase in rat muscle, and a second form in liver, brain, blood and other tissues. The study in rats did not detect an anodal brain isozyme analogous to that reported here in human brain. Enolase has been purified from several mammalian tissues, including muscle, brain, liver and erythrocytes (Wood, 1964; Baranowski, Wolna & Morawiecki, 1968; Witt & Witz, 1970; Rider & Taylor, 1974). In the single report describing the enzyme in brain tissue (Wood, 1964), the physical and chemical properties were very similar to those of enolase preparations from other tissues of various mammals. Human brain tissue appears to be the best source for the second locus product (i.e. band 111), although lower concentrations of this ,8p isozyme are present in other tissues, as well as in the cultured skin fibroblasts. It should therefore be possible to assign the second E N 0 locus to its chromosome by somatic cell hybridization (Ruddle, 1972). SUMMARY
Four different cellogel electrophoretic patterns of enolase were found in human tissue extracts. They consisted cf: (A) one strongly stained band (I)and two minor bands (I1and 111)found in haemolysates, white cell, skin fibroblast and kidney extracts ; (B) a three-banded pattern in brain resembling that of haemolysates except for heavier concentrations of bands I1 and 111; 18
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(C) a single component corresponding to band I, found in liver, heart, intestine, spleen and placenta ; (D) a single band with slightly faster mobility than band I, found in adult muscle extracts. The haemolysate of an individual with the heterozygous ENOilEN02, genotype had a triplebanded pattern replacing band I, a double-banded pattern replacing band I1 and a single weakly staining band 111.This finding, as well as the results of dissociation and recombination experiments, supports the hypothesis that the enzyme is a dimer formed by random interaction of two polypeptide chains (aand /3) synthesized by two independent gene loci (ENO, and ENO,). Evidence for a third locus, ENO,, is supplied by the electrophoretic pattern of muscle extracts. We thank Dr C. Ronald Scott, Department of Pediatrics, University of Washington, for supplying us adult post-mortem tissues and for reviewing the manuscript; and Dr Gilbert S. Omenn, Department of Medicine, University of Washington, for supplying some of the tissue extracts in this study. REFERENCES
BARANOWSKI, T., WOLNA, E. & MORAWIECIU, A. (1968). Purification and properties of crystalline a-phosphoD glycerate hydro-lyase from human muscle. Eur. J . Biochem. 5, 119. CIIEN, S.-H. & GIBLETT,E. R. (1971). Genetic variation of soluble glutamic-oxaloacetic transaminase in man. Am. J . Hum. Genet. 23,419. CHEN, 8.-H., SCOTT,C. R. & SWEDBERC, K. R. (1974). Heterogeneity of adenosine deaminase deficiency: expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells. Am. J . Hum. Genet. 27, 46.
GIBLETT, E. R., CHEN, S.-H., ANDERSON, J. E. & LEWIS, M. (1974). Afamily study suggesting genetic linkage of phospho-pyruvate hydratase (Enolase) to the Rh blood group system. Cytogenet. Cell Genet. 13, 91. HOLT, A. & WOLD,F. (1961). The isolation and characterization of rabbit muscle enolase. J . Biol. Chem. 236, 3227.
A. & WOSTERVELD,A. (1974). The human loci for MEERA KHAN,P., DOPPERT,B. A., HAOEMEIZER, phosphopyruvate hydratase and guanylate kinase are syntenic with the PGD-PGM, linkage groups in man-Chinese hamster somatic cell hybrids. Cytogenet. Cell Genet. 13, 130. PEARCE,J. M., EDWARDS, Y. H. & HARRIS,H. (1975). Human enolase isozyme: electrophoresis and biochemical evidence for three loci. Ann. Hum. Genet. 39, 363. RIDER,C. C. & TAYLOR,C. B. (1974). Enolase isozymes in rat tissues: electrophoretic chromatographic, immunological and kinetic properties. Bwchim. Biophys. Acta 365, 285. RUDDLE, F. H. (1972). In Advances i n Human Genetice 3 (ed. H. Harris and K. Hirschhorn). New York: Plenum Press. WESTHEAD, E. D. (1967). I n Methods i n Enzymology Z X (ed. S. P. Colowck), p. 670. New York: Academic Press. WITT, I. & WITZ, D. (1970). Reinigung und Charakterisierung von Phosphopyruvate-hydratase(enolase: E.C.4.2.1.11) aus Neugeborenen- und Erwachsenen-Erythrozyten. Zeit. PhySioZ. Chem. 351, 1232. WOLD,F. (1971). In The Enzymes I.' (ed. P. Boyer), p. 499. New York and London: Academic Press. WOOD, T. (1964). The purification of enolase from cerebral tissue. Biochm. J . 91, 453.
