Mutually Exclusive Splicing Generates Two Distinct Isoforms of Pig Heart Succinyl-CoA Synthetase
1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês
10.1074/jbc.272.34.21151
ISSN1083-351X
AutoresDavid G. Ryan, Tianwei Lin, Edward R. Brownie, William A. Bridger, William T. Wolodko,
Tópico(s)Plant biochemistry and biosynthesis
ResumoWe have identified two distinct cDNAs encoding the α-subunit of pig heart succinyl-CoA synthetase. The derived amino acid sequence of one of these, PHα57, is highly similar to the α-subunit of the rat liver precursor enzyme. The second cDNA, PHα108, was identical throughout its sequence with PHα57 except for a stretch of 108 nucleotides which replaced a 57 nucleotide sequence in PHα57. Coexpression of either α-subunit cDNA with a common pig heart β-subunit cDNA produced isozymes with GTP-specific enzyme activity. The enzyme produced by the combination of PHα57 and the β-subunit cDNA resembled the “native” enzyme purified from pig heart tissue. In contrast, the expressed enzyme from the combination with PHα108 was clearly distinguishable from the native enzyme by, for example, hydroxyapatite chromatography. Moreover, it was now apparent that this isoform had been observed in previous preparations of the native enzyme, but always in very low amounts and, thus, disregarded. We have shown further that the two mRNA transcripts arise from a single gene and are generated by mutually exclusive splicing. The production of the PHα108 message involves the use of a non-canonical splice site pair, AT-AA. Finally, we provide evidence for tissue specific regulation in the splicing of the PHα108 message. We have identified two distinct cDNAs encoding the α-subunit of pig heart succinyl-CoA synthetase. The derived amino acid sequence of one of these, PHα57, is highly similar to the α-subunit of the rat liver precursor enzyme. The second cDNA, PHα108, was identical throughout its sequence with PHα57 except for a stretch of 108 nucleotides which replaced a 57 nucleotide sequence in PHα57. Coexpression of either α-subunit cDNA with a common pig heart β-subunit cDNA produced isozymes with GTP-specific enzyme activity. The enzyme produced by the combination of PHα57 and the β-subunit cDNA resembled the “native” enzyme purified from pig heart tissue. In contrast, the expressed enzyme from the combination with PHα108 was clearly distinguishable from the native enzyme by, for example, hydroxyapatite chromatography. Moreover, it was now apparent that this isoform had been observed in previous preparations of the native enzyme, but always in very low amounts and, thus, disregarded. We have shown further that the two mRNA transcripts arise from a single gene and are generated by mutually exclusive splicing. The production of the PHα108 message involves the use of a non-canonical splice site pair, AT-AA. Finally, we provide evidence for tissue specific regulation in the splicing of the PHα108 message. Succinyl-CoA synthetase (SCS) 1The abbreviations used are: SCS, succinyl-CoA synthetase (EC 6.2.1.4 and EC 6.2.1.5); NDP, purine ribonucleoside diphosphate; NTP, purine ribonucleoside triphosphate; PCR, polymerase chain reaction; 5′-RACE, rapid amplification of 5′ cDNA ends; PMSF, phenylmethylsulfonyl fluoride; UTR, untranslated region; TCR, translational coupling region; bp, base pair(s); kb, kilobase pair(s). catalyzes the substrate-level phosphorylation step of the citric acid cycle according to the following reaction (Reaction 1).SuccinylCoA+NDP+Pi⇌succinate+CoA+NTPREACTION1(where N denotes adenosine or guanosine). SCS has a heterologous quaternary structure consisting of two subunits designated α and β with approximate molecular weights (Mr) of 30–32 × 103 and 42 × 103, respectively (see Ref. 1Nishimura J.S. Adv. Enzymol. 1986; 58: 141-172PubMed Google Scholar for review). Catalysis proceeds via the intermediate transfer of a phosphoryl group to and from a conserved histidine residue within the α-subunit (2Bridger W.A. Biochem. Biophys Res. Comm. 1971; 42: 948-954Crossref PubMed Scopus (36) Google Scholar, 3Wang T. Jurasek L. Bridger W.A. Biochemistry. 1972; 11: 2067-2070Crossref PubMed Scopus (9) Google Scholar). The three-dimensional structure of the Escherichia coli enzyme has been determined (4Wolodko W.T. Fraser M.E. James M.N.G. Bridger W.A. J. Biol. Chem. 1994; 269: 10883-10894Abstract Full Text PDF PubMed Google Scholar) and provides a solid framework for the interpretation of many studies. Apart from the primary role of succinyl-CoA within the citric acid cycle, high levels of this intermediate must be maintained for continued utilization of ketone bodies (5Ottaway J.H. McMinn C.L. Biochem. Soc. Trans. 1979; 7: 411-412Crossref PubMed Scopus (7) Google Scholar). In mammals, ketone bodies such as acetoacetate and β-hydroxybutyrate are produced by the liver, and tissues such as heart muscle routinely derive much of their metabolic energy from oxidation of these compounds (6Ottaway J.H. McClellan J.A. Saunderson C.L. Int. J. Biochem. 1981; 13: 401-410Crossref PubMed Scopus (44) Google Scholar). Although within the citric acid cycle SCS catalyzes the conversion of succinyl-CoA to succinate, ketone body activation requires the reverse, the conversion of succinate to succinyl-CoA. Ottaway and co-workers (5Ottaway J.H. McMinn C.L. Biochem. Soc. Trans. 1979; 7: 411-412Crossref PubMed Scopus (7) Google Scholar, 6Ottaway J.H. McClellan J.A. Saunderson C.L. Int. J. Biochem. 1981; 13: 401-410Crossref PubMed Scopus (44) Google Scholar) have suggested that such opposing functional demands could best be accommodated by distinct forms of the enzyme. It is worth noting that succinyl-CoA also serves as an anabolic precursor in the synthesis of porphyrins and hemes (7Lascelles, J. (1964) in Tetrapyrrole Biosynthesis and Its Regulation, (Benjamin, W. A., Ed.) New. York., p. 84.Google Scholar). Historically, there is evidence that diverse isoforms of the mammalian enzyme exist. Baccanari and Cha (8Baccanari D.P. Cha S. J. Biol. Chem. 1973; 248: 15-24Abstract Full Text PDF PubMed Google Scholar) showed that the GTP-specific enzyme from pig heart tissue separated into several species differing in charge. These multiple forms were found to be interconvertible indicating a common protein source (9Baccanari D.P. Cha S. Biochim. Biophys. Acta. 1973; 119: 226-234Google Scholar). In a different study, two GTP-specific forms of pig heart SCS were separated upon purification using hydroxyapatite chromatography (10Cha S. Cha C.-J.M. Parks Jr., R.E. J. Biol. Chem. 1967; 242: 2577-2581Abstract Full Text PDF PubMed Google Scholar). A similar observation has been reported of the enzyme isolated from pigeon pectoral muscle (11Meshkova N.P. Matveeva L.N. Biokhimiya. 1970; 35: 374-381PubMed Google Scholar). Two distinct GTP-specific forms of succinyl-CoA synthetase were detected in mouse liver, one of which was induced as a result of increased porphyrin synthesis (12Labbe R.F. Kurumada T. Onisawa J. Biochim. Biophys. Acta. 1965; 111: 403-415Crossref PubMed Scopus (36) Google Scholar). More recently, Weitzman et al. (13Weitzman P.D.J. Jenkins T. Else A.J. Holt R.A. FEBS Lett. 1986; 199: 57-60Crossref PubMed Scopus (18) Google Scholar) demonstrated the existence of isoforms differing in their nucleotide specificity in mammalian tissues. Separable GTP- and ATP-specific forms were present in ratios that varied depending on the tissue source. Marked increases in the GTP-specific form in brain tissue occur following treatment of rats with streptozotocin, a drug used to induce diabetes (14Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1986; 205: 215-218Crossref PubMed Scopus (22) Google Scholar). Diabetes invariably leads to elevated levels of ketone bodies. These and other observations have led to the suggestion that a GTP-specific form is involved exclusively in ketone body metabolism (14Jenkins T.M. Weitzman P.D.J. FEBS Lett. 1986; 205: 215-218Crossref PubMed Scopus (22) Google Scholar, 15McClellan J.A. Ottaway J.H. Comp. Biochem. Physiol. 1980; 67B: 679-684Google Scholar). The focus of the studies described herein was to gain a better understanding of one source of the heterogeneity present with the pig heart enzymes. Here we report on the identification and characterization of two isoforms of the α-subunit of the pig heart enzyme. The two forms are generated by alternative splicing of a single transcript. Furthermore, we provide evidence for tissue-specific regulation of the splicing event. The following E. coli strains were used: JM109 (16Yanish-Perron C. Viera J. Messing J. Gene. 1985; 33: 103-199Crossref PubMed Scopus (11461) Google Scholar) for construction and propagation of M13 and plasmid derivatives, BL21(DE3) (17Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-119Crossref PubMed Scopus (4831) Google Scholar) for expression studies, and Y1089 (18Young R. Davis R.W. Science. 1983; 222: 778-789Crossref PubMed Scopus (602) Google Scholar) and LE392 (19Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual.1st Ed. Cold Spring Harbor Laboratory Press, 1982Google Scholar) for screening of cDNA and genomic libraries, respectively. The polynucleotide kinase, ligase, Klenow DNA polymerase, and restriction enzymes were obtained from Life Technologies (as Gibco BRL products) and New England Biolabs Inc. The Taq DNA polymerase was purchased from Cetus Corp. Reagents used for sequencing nucleic acids were ordered from U. S. Biochemical Corp. The dNTPs were purchased as 100 mmsolutions from Pharmacia Biotech Inc. Unless otherwise specified, all other chemicals were supplied by Sigma and BDH Chemicals Ltd. The generation of the pig heart cDNA library used in this study has been reported elsewhere (20Lin T. Bridger W.A. J. Biol. Chem. 1992; 267: 975-978Abstract Full Text PDF PubMed Google Scholar). The unamplified library was screened with a biotin-labeled probe prepared from a cDNA encoding the precursor α-subunit of rat liver SCS (21Henning W.D. Upton C. Majumdar R. McFadden G. Bridger W.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1432-1436Crossref PubMed Scopus (21) Google Scholar). Potential clones were identified with streptavidin-conjugated alkaline phosphatase (22Huynh T.V. Young R.A. Davis R.W. Glover D.M. DNA Cloning. IRL Press Limited, Oxford1985Google Scholar, 23Haas M.J. Fleming D.J. Anal. Biochem. 1988; 168: 239-242Crossref PubMed Scopus (8) Google Scholar). The cDNA fragments were subcloned into M13 vectors (16Yanish-Perron C. Viera J. Messing J. Gene. 1985; 33: 103-199Crossref PubMed Scopus (11461) Google Scholar), and their sequences were determined by the dideoxynucleotide chain termination method (24Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52650) Google Scholar). High molecular mass DNA was prepared from the liver of a mature pig (25Blin N. Stafford W.D. Nucleic Acids Res. 1976; 3: 2303-2308Crossref PubMed Scopus (2332) Google Scholar), and used to construct a genomic library in λGEM11 following standard procedures (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, 1989Google Scholar). The λGEM11 arms and packaging extracts were bought from Promega Corp. A second genomic library, constructed in λEMBL-3, was purchased from CLONTECHLaboratories. Both genomic libraries (1 × 106 plaques each) were screened under conditions of high stringency (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, 1989Google Scholar) with the cDNA clone, PHα57. This probe was radioactively labeled with [α35S]dATP (Amersham Canada Ltd.) by random priming (27Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16653) Google Scholar). Southern blot analyses were carried out using standard methods with GeneScreen membranes (DuPont NEN) and oligonucleotide probes end-labeled with 32P. Total RNA was purified from fresh tissues of newborn piglets (generously provided by Dr. G. Foxcroft, Faculty of Agriculture, Forestry, and Home Economics). Trizol reagent (Life Technologies) was used to purify the RNA. An aliquot of RNA (1 μg) was reverse transcribed with SuperScript II (Life Technologies) and oligo(dT). Reverse transcribed PCR for each isoform was carried out pairing oligonucleotide 6, below, with either oligonucleotide 3 (specific for PHα57) or oligonucleotide 5 (specific for PHα108) as the primers. The cycling conditions used were as follows: 20 s at 94 °C, 30 s at 57 °C, and 60 s at 68 °C (40 cycles for PHα57, 45 cycles for PHα108, and 35 cycles for β-actin); followed by one cycle of 10 min at 72 °C. The PCR was performed as described by Saiki et al. (28Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13489) Google Scholar). The procedures of Frohman et al. (29Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8995-8999Google Scholar) were followed for the 5′-RACE analyses using the appropriate reagents purchased from Life Technologies. RNA (1 μg) was reverse transcribed with SuperScript II and oligonucleotide 2, below, as the primer. This primer was used for first strand synthesis of specifically α-subunit cDNAs carrying either the 57 or the 108 nucleotide sequence. These cDNAs were purified through a GlassMax spin cartridge (Life Technologies) and were tailed with dCTP and TdT. Anchored PCR was carried out with a 5′-RACE anchor primer and oligonucleotides specific for PHα57 ( oligonucleotide 3 below) and PHα108 (oligonucleotide 5 below). The cycling conditions were as follows: 42 cycles of 20 s at 94 °C, 30 s at 57 °C, and 60 s at 68 °C; followed by one cycle of 10 min at 72 °C. The amplified products were purified and cloned directly into TA vectors (Invitrogen Corporation). The sequences of the oligonucleotides used in all these studies are as follows.1:2:3:4:5:6:*CACAGGCTGCTGCGCCAGGGAAA*CCAACTTGTGTTGTTTGATGAACTGC*TGAATATGGCCAGGCATGATG*GTGCATGCTGGAACCAGAAGTA*CTTCCTGTATTCTTCCTGCTG*GAAGGTTATTTGCCAGGGTTTCAC The plasmid, pT7-6 (30Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2455) Google Scholar) was used to construct vectors capable of co-producing both subunits of SCS. The configuration of the recombinant vector, pT7-6Ecβ/Ecα, carrying the genes for the E. coli enzyme is illustrated in Fig. 3 A. Both cDNAs of the pig heart α-subunits were modified to include aClaI restriction site at the equivalent position relative to their bacterial counterpart. These modified sequences were cloned into pT7-6 Ecβ/Ecα in place of the E. coli gene. The resulting plasmids, pT7-6 Ecβ/PHα57 and Ecβ/PHα108, were used as negative controls; although the expression of the individual E. coli β-subunit and the pig heart α-subunit can occur, the subsequent formation of SCS hybrids does not. A cDNA sequence encoding the pig heart β-subunit (31Bailey D.L. Wolodko W.T. Bridger W.A. Protein Science. 1993; 2: 1255-1262Crossref PubMed Scopus (16) Google Scholar) was altered to include NdeI andEspI restriction sites at the appropriate locations. TheE. coli gene coding for the β-subunit in pT7-6Ecβ/PHα57 and Ecβ/PHα108 was replaced with the modified pig heart β-subunit sequence creating the expression plasmids, pT7-6 PHβ/PHα57 and pT7-6 PHβ/PHα108. The above mentioned sequence alterations were achieved using standard mutagenesis procedures (32Zoller M.J. Smith M. Meth. Enzymol. 1983; 100: 468-500Crossref PubMed Scopus (661) Google Scholar) on uracil-enriched templates (33Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). The mutagenic oligonucleotides were as follows.ClaI:EspI:NdeI:*CATCTCTATATCGATAAAAATACG*GGCCACAGCCTGCTGAGCTGCATCCTC*AGACTGAACATATGAACCTGCAGGAFor protein expression, typically 2.5-liter cultures of BL21(DE3) carrying the expression plasmids were grown from freshly transformed cells and were incubated at 37 °C until theA600 reached a value of 0.6. Induction of expression was achieved by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm. Following induction, the cultures were allowed to incubate further at 37 °C for 5 h with vigorous agitation, after which the cells were harvested and frozen. The frozen bacterial cultures were thawed and resuspended in 100 ml of “sonication” buffer (50 mm potassium phosphate, pH 7.4, 1 mm EDTA, 0.1 mm PMSF). These suspensions were sonicated over crushed ice and centrifuged to pellet the cell debris. Each supernatant was subjected to a two-step ammonium sulfate precipitation. The protein fraction precipitating between 15% and 40% (w/v) ammonium sulfate was collected by centrifugation and redissolved in a minimal volume of 10 mm potassium phosphate, 1 mm EDTA, 0.1 mm PMSF at pH 7.4. These solutions were desalted by gel filtration through Sephadex G25 equilibrated and eluted with the same dissolving buffer. Eluted fractions that contained high enzyme activity were pooled and loaded directly onto a 50-ml hydroxyapatite column equilibrated with 10 mm potassium phosphate, pH 7.4, 1 mm EDTA, 0.1 mm PMSF. The column was washed with another 200 ml of the buffer. Two columns were set up and run simultaneously in this manner, one for expressed PHβ/PHα57 and the other for expressed PHβ/PHα108. Elution (from a common 500-ml gradient reservoir) was carried out with a salt gradient of 10 mm to 400 mm potassium phosphate, pH 7.4. All of the above purification procedures were conducted at 4 °C. Protein extracts made from minced pig heart tissue were fractionated in a similar manner. Succinyl-CoA synthetase activity was measured by following the change of absorbance at 235 nm using the method of Cha (34Cha S. Meth. Enzymol. 1969; 13: 62-69Crossref Scopus (46) Google Scholar). Approximately 105 plaques from an unamplified pig heart cDNA library were screened as described under “Materials and Methods.” A total of 25 clones were recovered from the library. Of these, 12 were found to be full-length and were selected for further analysis. It was observed that for all these clones two fragments were produced upon EcoRI digestion, indicative of a single, internal EcoRI site. The clones were classified into two groups on the basis of the size of the largerEcoRI fragment (Fig. 1 A). Class I accounted for nine of these clones and contained a 1.15-kb EcoRI fragment. The remaining Class II clones contained a slightly larger 1.2-kb fragment. Representatives from each group were selected for sequencing. The two classes of cDNAs were identical throughout their sequence, with the exception of one short region midway through their open reading frames (Fig. 1 A). A stretch of 108 nucleotides in the Class II clones replaced a 57-nucleotide sequence in the Class I clones.Figure 1Sequences of cDNAs encoding the α-subunit of pig heart succinyl-CoA synthetase. A, diagrammatic alignment of Class I and II cDNAs on the basis of sequence identity (thick lines). The arrowsindicate open reading frames. The boxes highlight the regions of sequence differences: clear box, 57 bp;striped box, 108 bp. Given in B is the complete nucleotide sequence of a representative Class I clone, PHα57. The derived amino acid sequence of the α-subunit of pig heart succinyl-CoA synthetase is shown below in single-letter code. The vertical arrow shows the cleavage point of the signal sequence. The numerals on the right(in bold) count the amino acids and on the left(in italics) the nucleotides. As in A, the 57 nucleotides specific to Class I clones are outlined by abox. Shown in C is the corresponding region of a Class II clone, PHα108, highlighting the unique 108 nucleotides in abox.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The nucleotide and deduced amino acid sequence of a Class I clone, PHα57, is shown in Fig. 1 B. The cDNA PHα57 encodes a 333-amino acid protein with a high degree of similarity to the precursor α-subunit of the rat liver enzyme (21Henning W.D. Upton C. Majumdar R. McFadden G. Bridger W.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1432-1436Crossref PubMed Scopus (21) Google Scholar): 305 of the 333 amino acids are identical in the two subunits. Both mammalian proteins carry a putative signal peptide of 27 amino acids, which would be removed upon entry into the mitochondria (35Majumdar R. Bridger W.A. Can. J. Biochem. Cell Biol. 1990; 68: 292-299Crossref Scopus (6) Google Scholar). Part of the sequence derived from a Class II clone, PHα108, is shown in Fig. 1 C. As stated previously, PHα108 was identical to PHα57 except for the replacement of a short stretch of sequence within its open reading frame. Substitution of this sequence preserves the reading frame and results in a novel 36 amino acid stretch within the α-subunit. The identities of the remaining clones were designated using the presence of a unique NdeI site at the 3′ end of the PHα108-specific sequence (Fig. 1 A) as a discriminating characteristic of Class II clones. The molecular masses of the mature proteins (without signal sequence) encoded by PHα57 and PHα108, were calculated to be 32.11 kDa and 34.45 kDa, respectively. The value predicted for the PHα57-encoded protein is consistent with that estimated for the α-subunit of “native” SCS purified from pig heart tissue (36Brownie E. Bridger W.A. Can. J. Biochem. 1972; 50: 719-724Crossref PubMed Scopus (16) Google Scholar). Expression of PHα57 in bacteria produced a protein that co-migrated with the α-subunit of the native enzyme on SDS-polyacrylamide gel electrophoresis, whereas PHα108 produced a significantly larger protein. Amino acid analyses of peptide fragments generated by cyanogen bromide treatment of the native enzyme identified the α-subunit encoded by PHα57 as that corresponding to the subunit present in native enzyme. Moreover, the amino acid sequence derived from the 57 nucleotides present in the PHα57 cDNA is conserved in known forms of SCS (Fig. 2 A). The unique sequence from PHα108 was not found in a search of the various data banks. The crystallographic model of the α-subunit of the E. colienzyme (illustrated in Fig. 2 B) was examined focusing on the corresponding location of the unique segments encoded by PHα57 and PHα108. The amino acid sequence replaced in the two pig heart isoforms corresponds to a polypeptide stretch that interconnects the two domains of the α-subunit: the CoA-binding domain and the phosphohistidine domain (Fig. 2 B). Note: as can be seen in Fig. 2 A, the amino acid residues are highly conserved on either side of both interconnecting sequences and are thought to play a role in catalysis. The sequence SRSGTLTYE to the right forms an α-helix that stabilizes the phosphohistidine loop, whereas the residues to the left come in close contact with the reactive thiol group of CoA (4Wolodko W.T. Fraser M.E. James M.N.G. Bridger W.A. J. Biol. Chem. 1994; 269: 10883-10894Abstract Full Text PDF PubMed Google Scholar). This comparison to the model of the E. coli α-subunit serves to demonstrate that the longer amino acid sequence encoded by PHα108 can be accommodated within the structure of the protein. Prompted by the earlier reports of heterogeneity in the enzyme (10Cha S. Cha C.-J.M. Parks Jr., R.E. J. Biol. Chem. 1967; 242: 2577-2581Abstract Full Text PDF PubMed Google Scholar, 11Meshkova N.P. Matveeva L.N. Biokhimiya. 1970; 35: 374-381PubMed Google Scholar, 12Labbe R.F. Kurumada T. Onisawa J. Biochim. Biophys. Acta. 1965; 111: 403-415Crossref PubMed Scopus (36) Google Scholar, 13Weitzman P.D.J. Jenkins T. Else A.J. Holt R.A. FEBS Lett. 1986; 199: 57-60Crossref PubMed Scopus (18) Google Scholar), we considered both pig heart α-subunits as authentic proteins contributing to enzyme activity. To better understand the functional significance of the novel PHα108-encoded protein, a bicistronic expression system in bacteria was developed for the production of the pig heart enzymes. The recombinant vector, pT7-6Ecβ/Ecα (Fig. 3 A), had been used previously for the production of the E. coli enzyme (41Ryan D.G. Bridger W.A. J. Mol. Biol. 1991; 219: 165-174Crossref PubMed Scopus (3) Google Scholar). In this vector, the two genes for the E. coli enzyme overlap in such a manner that translation of the α-subunit is coupled to that of the β-subunit, ensuring that equal levels of both are produced (37Buck D. Spencer M.E. Guest J.R. Biochemistry. 1985; 24: 6245-6252Crossref PubMed Scopus (55) Google Scholar). The nucleotide sequence responsible for this coupling is referred to here as the translational coupling region (TCR) and occurs within the overlapping ends of the two genes as illustrated in Fig. 3A. The β-subunit of SCS from either E. coli or pig heart was produced in combination with each α-subunit isoform (see “Materials and Methods”). The TCR was retained in all of these cDNA constructs (Fig. 3 B). Although high levels of the subunits were produced by expression, greater than 90% of this protein was insoluble. Despite this, we could clearly measure elevated levels of a GTP-specific activity in the bacterial lysates following the expression of both PHβ/PHα57 and PHβ/PHα108. The enzyme of the hostE. coli cells uses ATP or GTP as the nucleotide substrate (42Murakami K. Mitchell T. Nishimura J.S. J. Biol. Chem. 1972; 247: 6247-6252Abstract Full Text PDF PubMed Google Scholar), contributing a basal SCS activity in these experiments. As shown in Fig. 3 C, when measured from the time of induction to 5 h, there was little increase above basal levels of the ATP- or GTP-specific activities by the controls (Ecβ/PHα57 andEcβ/PHα108). In contrast, the GTP-specific activity associated with the expression of the two plasmids PHβ/PHα57 and PHβ/PHα108 increased by a factor of two over the course of the experiment, while the ATP-specific activity remained low and relatively constant as seen in the controls. We conclude that two separate isoenzymes with GTP-specific activities are produced by the combination of each of the two α-subunit isoforms with the pig heart β-subunit. It was first reported by Cha et al. (10Cha S. Cha C.-J.M. Parks Jr., R.E. J. Biol. Chem. 1967; 242: 2577-2581Abstract Full Text PDF PubMed Google Scholar) that conventional preparations of SCS from pig heart tissue were resolved into two separate peaks of GTP-specific activity by hydroxyapatite column chromatography. We have consistently made the same observation during routine purification of the native pig heart enzyme. A profile monitoring the GTP-specific activity of one such purification is presented in Fig. 4 C. The first peak of enzymatic activity to emerge from the column is never more than 10% of the total, but is consistently present and invariably discarded. The second peak to emerge often accounts for greater that 90% of the total enzyme; these fractions are pooled for further purification. In the present studies, bacterial cell extracts from cultures with the two expression combinations PHβ/PHα57 and PHβ/PHα108 were prepared for hydroxyapatite column chromatography as described under “Materials and Methods.” Their simultaneous fractionation on identical columns is illustrated in Fig. 4(A and B, respectively). The first peak of enzymatic activity that emerged from both columns with 65 mm phosphate was specific for both ATP and GTP, a characteristic of the host bacterial SCS. The second peak of activity detected, albeit at different points of elution in both fractionations, showed an absolute specificity for GTP, that expected of the pig heart enzymes. As can be seen by comparing Fig. 4 A with Fig. 4 C, the GTP-specific activity produced by the PHβ/PHα57 combination eluted from the column in the range of 170 mmphosphate, coincident with the main peak of a native pig heart SCS preparation. It is not surprising, then, that the native enzyme purified from pig heart tissue had been found to comprise the PHα57 protein. It was, however, a surprise to find that the GTP-specific activity produced by the PHβ/PHα108 combination emerged earlier with 110 mm phosphate, colinear with the minor peak of a native preparation (compare Fig. 4 B with Fig. 4 C). Unfortunately, attempts to purify this minor isozyme from the others in pig heart tissue have met with limited success. Further experiments were carried out with the aim of delineating the molecular mechanisms involved in generating these two nearly identical cDNAs. We screened two pig genomic DNA libraries (see “Materials and Methods”). Two clones, designated λE4 and λE6, were recovered from a λEMBL-3 library. Fragments of λE6 hybridized to a probe specific for the 57-nucleotide sequence (oligonucleotide 3 under “Materials and Methods”) but not to a probe specific for the 108-nucleotide sequence (oligonucleotide 4). In contrast, fragments of λE4 hybridized only to the probe specific for the 108-nucleotide sequence. The positive hybridization results are presented in Fig. 5 A. Furthermore, it appeared that these clones represented two parts of the α-subunit gene since λE6 also contained the remaining upstream sequence while λE4 contained the remaining downstream sequence of the α-subunit. The two clones failed to hybridize to each other, and thus did not overlap. A third clone, λ3551, was recovered from a λGEM11 library and proved to overlap both λE6 and λE4. As shown by Southern blot analyses, an 11-kb fragment of λ3551 hybridized to both probes (Fig. 5 A). This clearly established that the two isoform sequences originate from within a common gene. A map generated from extensive trials of restriction digestions and hybridization analyses of these clones has been compiled in Fig. 5 B. The sizes of the relevant introns were estimated using methods involving PCR (see Fig. 5 B). The intron upstream of the 57-nucleotide sequence was 200 bp, and the intron downstream of the 108-nucleotide sequence was 1.3 kb. PCR carried out directly on genomic DNA with the
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