- Email: [email protected]
glyoxylate reductase
Biochimica et Biophysica Acta 1446 (1999) 383^388 www.elsevier.com/locate/bba
Short sequence-paper
Identi¢cation and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase G. Rumsby *, D.P. Cregeen Department of Molecular Pathology, University College London, Windeyer Institute of Medical Sciences, Cleveland St, London W1P 6DB, UK Received 26 March 1999; received in revised form 4 June 1999; accepted 17 June 1999
Abstract The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame of 987 bp with a 225-bp 3P-untranslated region. The 328-amino acid protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial species. ß 1999 Elsevier Science B.V. All rights reserved.
Two inherited disorders which cause endogenous overproduction of oxalate have been described in the literature. Both diseases lead to recurrent renal stones and, in the most severe cases, to end stage renal failure and death. Primary hyperoxaluria type 1 (PH1) is caused by decreased activity of alanine: glyoxylate aminotransferase, an hepatic peroxisomal enzyme. Primary hyperoxaluria type 2 (PH2) is less well documented but is characterised by hyperoxaluria and the presence of elevated L-glycerate in the urine. Metabolic studies suggested that the underlying defect in PH2 could be explained by reduced cytosolic hydroxypyruvate reductase (HPR) activity [1], with the excess hydroxypyruvate reduced to L-glycerate in a reaction catalysed by lactate dehydrogenase (LDH; EC 1.1.1.27). HPR also has D-glycerate dehydrogenase (D-GDH; EC 1.1.1.29) [2] and glyoxylate reductase (GR; EC 1.1.1.26/79)
* Corresponding author. Fax: +44-171-504-9496; E-mail: [email protected]
[3] activities. It is now believed that the enzyme functions primarily as a reductase rather than a dehydrogenase [3,5] producing the gluconeogenic precursor D-glycerate from hydroxypyruvate and acting as a mechanism to remove the highly reactive oxalate precursor glyoxylate from the cytosol thus preventing its conversion to oxalate (Fig. 1). Tissue distribution studies suggest that there is more than one enzyme with hydroxypyruvate reductase activity in humans [6] and we have recently demonstrated that human liver HPR can be fractionated by chromatofocusing into two peaks di¡ering in their isoelectric points and substrate a¤nities [7]. Only one of these forms reduces both hydroxypyruvate and glyoxylate when NADPH is used as cofactor and is presumed to be the enzyme lacking in PH2. This paper o¡ers the ¢rst full description of a gene encoding human HPR/GR and will provide the means to elucidate the role of this gene product in the metabolism of oxalate precursors as well as enabling the molecular diagnosis of PH2. The Entrez database (http://www.ncbi.nlm.nih. gov/htbin-post/Entrez/) was screened using the term
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 0 5 - 0
BBAEXP 91286 18-8-99
384
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
Fig. 1. Proposed pathway of glyoxylate and hydroxypyruvate metabolism in mammalian liver. AGT, alanine:glyoxylate aminotransferase; HPR, hydroxypyruvate reductase; GR, glyoxylate reductase; GO, glycolate oxidase.
`hydroxypyruvate reductase' and a cDNA identi¢ed with peptide sequence homology to HPR from Hyphomicrobium methylovorum. The DNA sequence of this cDNA was used to screen the EST database for clones containing additional 5P-sequence and the longest cDNA clone obtained from the IMAGE Consortium [LLNL] (web site http://bbrp.llnl.gov/bbrp/ image) [8] and sequenced in full. The full-length cDNA clone (IMAGE Consortium clone ID:503200) was ampli¢ed by polymerase chain reaction (PCR) using oligonucleotide primers (Genosys Biotechnologies, Pampisford, UK) designed to introduce restriction sites for KpnI and HindIII at the 5P- and 3P-ends, respectively (forward primer, 5P-ATGGTACCGGGTCGGCGGCTG; reverse primer, 5P-GCAAGCTTCCCTTGGCTCTGC). PCR conditions were as follows: 5 ng DNA, 50 mmol/l potassium chloride, 10 mmol/l Tris HCl, pH 9.0, 0.1% Triton X-100, 2 mmol/l magnesium chloride, 200 Wmol/l dNTP, 0.6 Wmol/l each primer, 0.25 U Taq polymerase (Promega, Southampton, UK) in a volume of 25 Wl. Thirty cycles of 94³C 0.5 min, 66³C 0.5 min, 72³C for 1.5 min were carried out. Following digestion with KpnI and HindIII, the PCR product was ligated into the pTrcHis B expres-
sion vector (Invitrogen, The Netherlands) which had been cut with the same enzymes and the cut ends dephosphorylated. The cDNA was inserted into the multiple cloning site downstream of the His tag and Anti-Xpress antibody epitope and in frame with the plasmid initiation codon. The construct (pTrcHisBHPR) was transfected into Epicurean coli BL21 (DE3) competent cells (Stratagene, Cambridge, UK). Individual colonies were cultured overnight in SOB medium (20 g/l tryptone, 5 g/l yeast extract, 0.5 g/l sodium chloride, 2.5 mmol/l potassium chloride, 10 mmol/l magnesium chloride pH 7.0) containing ampicillin at a ¢nal concentration of 50 Wg/ml. Following plasmid puri¢cation, digestion with KpnI and HindIII con¢rmed the presence of an insert of the correct size (1093 bp). Single colonies of pTrcHisB and pTrcHisB-HPR in BL21 cells were grown up overnight in 2 ml SOB medium plus ampicillin (50 Wg/ml). Aliquots (50 ml) of SOB medium containing ampicillin (50 Wg/ml) were inoculated with 0.2 ml of the overnight cultures and cells grown with aeration to an absorbance at 600 nm of 0.6. Isopropylthio-L-D-galactoside (¢nal concentration 1 mmol/l) was added and the culture incubated for a further 5 h with shaking. The cells were spun down (3000 rpm for
BBAEXP 91286 18-8-99
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
385
Fig. 2. cDNA and predicted protein sequence of human HPR/GR (GLXR). Putative polyA addition signal sequence (AATAA) is underlined. Sequence deposited in GenBank submission no. AF134895.
BBAEXP 91286 18-8-99
386
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
10 min), washed once with PBSA, pelleted again and the pellet sonicated on ice in 1 ml 100 mmol/l potassium phosphate bu¡er, pH 8.0 containing 240 mmol/l sucrose using three, 10-s bursts from a Microson XL sonicator. The cell debris was pelleted by centrifugation at 13 000 rpm for 10 min at 4³C. HPR and GR activity was determined in the supernatant as previously described [6]. Six Wg of total cell protein from sonicate supernatants were electrophoresed in rehydrated polyacrylamide gels (Clean gel 36S, Amersham Pharmacia Biotech, St Albans, UK). After blotting onto nitrocellulose, non-speci¢c binding sites were blocked by immersion in 3% (w/v) milk proteins in PBSA, followed by incubation of the blot overnight with
Anti-Xpress antibody (Invitrogen) at a ¢nal dilution of 1 in 5000. Excess antisera was removed by washing in PBSA (2U10 min) and the blot incubated with alkaline phosphatase conjugated, goat-antimouse IgG (Sigma, Poole) for 3 h at room temperature. After two, 10-min washes in PBSA, the blot was immersed in alkaline phosphatase Colour Development reagent (Bio-Rad, Welwyn Garden City) for 25 min. The cDNA identi¢ed from the IMAGE library is 1235 bp, with 41 bp of 5P-untranslated region and an open reading frame of 987 bp (Fig. 2). The gene encoded by this cDNA has been assigned the symbol GLXR by the Nomenclature Committee of the Human Genome Project. The 3P-untranslated region is
Fig. 3. Peptide sequence of human HPR/GR aligned with glycerate dehydrogenase (NADH-dependent HPR/GR) from Hyphomicrobium methylovorum (hypho) (GenBank P36234), Cucumis sativus (cucurbit) (Genbank P13443) and Methylobacterium extorquens (methylo) (Genbank Q59156).
BBAEXP 91286 18-8-99
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
387
Fig. 4. HPR (shaded boxes) and GR (open boxes) activity in BL21 cells transfected with pTrcHisB-HPR and pTrcHisB, respectively.
225 bp with a putative polyadenylation signal sequence (AATAA) 14 nucleotides from the end of the cDNA. The sequence is predicted to encode a protein of 328 amino acids with a molecular weight of 36.5 kDa (http://www.expasy.ch/tools/pi-tool. html). The protein has sequence similarity with HPR/GR from H. methylovorum (32%), Cucucmis sativus (32%) and Methylobacterium extorquens (21%) (Fig. 3). Expression of the cDNA in BL21 bacterial cells produced a protein with GR and HPR activities of 631 þ 22 and 509 þ 17 nmol NADPH oxidised/min/ mg protein respectively (mean þ 1 S.D. from six analyses) compared with activities of 124 þ 7 nmol/min/ mg protein and 16 þ 8 nmol/min/mg in cells transfected with vector alone (Fig. 4). A fusion protein of 43 kDa was detected on Western blot analysis which was not present in sonicates of cells transfected with vector alone. As the fusion protein adds an additional 3 kDa to the protein size, this result indicates that the protein translated from the cDNA is approximately 40 kDa, slightly bigger than the predicted 36.5 kDa predicted from the amino acid sequence. The same size band was detected under denaturing and non-denaturing conditions suggesting that the enzyme is present as a monomer and is active in this form. In the present study, we have identi¢ed a fulllength cDNA clone from human liver by virtue of its homology to HPR from H. methylovorum. Ex-
pression of the cDNA in BL21 cells produced a fusion protein of 43 kDa with the ability to reduce both glyoxylate and hydroxypyruvate in the presence of NADPH as cofactor. As the plasmid transcript provides an additional 40 amino acids, the actual size of the expressed HPR/GR protein is of the order of 40-kDa. The deduced protein sequence shares approximately 30% sequence similarity with HPR and GR from a number of species of plants and bacteria including H. methylovorum, C. sativus and M. extorquens (Fig. 3). There was no signi¢cant similarity with any of the mammalian lactate dehydrogenase genes. Analysis of the peptide sequence using the Prosite program [9] revealed a 2-hydroxyacid dehydrogenase signature starting at codon 232 (MKETAVFINISRGDVVN). A cDNA of similar size encoding a protein with D-GDH activity has been described in abstract form [10], but no information was given as to its activity with hydroxypyruvate or glyoxylate or its nucleotide or peptide sequence and therefore no further comparisons are possible. However, in view of the fact that GR, HPR and D-GDH activities are all decreased in PH2 [1,4] and therefore that one enzyme appears to have all three activities, it is possible that the two cDNA species are the same. Further studies will elucidate the kinetics of this enzyme, physical properties and role in metabolism. The discovery of this gene also enables the molecular genetics of PH2 to be established.
BBAEXP 91286 18-8-99
388
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
This project was funded by a grant from the Oxalosis and Hyperoxaluria Foundation. References [1] H. Williams, L.J. Smith, New Engl. J. Med. 278 (1968) 233^ 239. [2] J.E. Willis, H.J. Sallach, J. Biol. Chem. 237 (1962) 910^915. [3] P. Dawkins, F. Dickens, Biochem. J. 94 (1965) 353. [4] J. Mistry, C.J. Danpure, R.A. Chalmers, Biochem. Soc. Trans. 16 (1988) 626^627.
[5] E. Van Schaftingen, J.-P. Draye, F.V. Van Hoof, Eur. J. Biochem. 186 (1989) 355^359. [6] C.F. Gia¢, G. Rumsby, Ann. Clin. Biochem. 35 (1998) 104^ 109. [7] D. Cregeen, G. Rumsby. J. Am. Soc. Nephrol., in press. [8] G.G. Lennon, C. Au¡ray, M. Polymeropoulos, M.B. Soares, Genomics 33 (1996) 151^152. [9] A. Bairoch, P. Bucher, K. Hofmann, Nucleic Acids Res. 25 (1997) 217^221. [10] S.D. Cramer, K. Lin, R.P. Holmes, J. Urol. 159 (5S) (1998) 661.
BBAEXP 91286 18-8-99
Short sequence-paper
Identi¢cation and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase G. Rumsby *, D.P. Cregeen Department of Molecular Pathology, University College London, Windeyer Institute of Medical Sciences, Cleveland St, London W1P 6DB, UK Received 26 March 1999; received in revised form 4 June 1999; accepted 17 June 1999
Abstract The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame of 987 bp with a 225-bp 3P-untranslated region. The 328-amino acid protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial species. ß 1999 Elsevier Science B.V. All rights reserved.
Two inherited disorders which cause endogenous overproduction of oxalate have been described in the literature. Both diseases lead to recurrent renal stones and, in the most severe cases, to end stage renal failure and death. Primary hyperoxaluria type 1 (PH1) is caused by decreased activity of alanine: glyoxylate aminotransferase, an hepatic peroxisomal enzyme. Primary hyperoxaluria type 2 (PH2) is less well documented but is characterised by hyperoxaluria and the presence of elevated L-glycerate in the urine. Metabolic studies suggested that the underlying defect in PH2 could be explained by reduced cytosolic hydroxypyruvate reductase (HPR) activity [1], with the excess hydroxypyruvate reduced to L-glycerate in a reaction catalysed by lactate dehydrogenase (LDH; EC 1.1.1.27). HPR also has D-glycerate dehydrogenase (D-GDH; EC 1.1.1.29) [2] and glyoxylate reductase (GR; EC 1.1.1.26/79)
* Corresponding author. Fax: +44-171-504-9496; E-mail: [email protected]
[3] activities. It is now believed that the enzyme functions primarily as a reductase rather than a dehydrogenase [3,5] producing the gluconeogenic precursor D-glycerate from hydroxypyruvate and acting as a mechanism to remove the highly reactive oxalate precursor glyoxylate from the cytosol thus preventing its conversion to oxalate (Fig. 1). Tissue distribution studies suggest that there is more than one enzyme with hydroxypyruvate reductase activity in humans [6] and we have recently demonstrated that human liver HPR can be fractionated by chromatofocusing into two peaks di¡ering in their isoelectric points and substrate a¤nities [7]. Only one of these forms reduces both hydroxypyruvate and glyoxylate when NADPH is used as cofactor and is presumed to be the enzyme lacking in PH2. This paper o¡ers the ¢rst full description of a gene encoding human HPR/GR and will provide the means to elucidate the role of this gene product in the metabolism of oxalate precursors as well as enabling the molecular diagnosis of PH2. The Entrez database (http://www.ncbi.nlm.nih. gov/htbin-post/Entrez/) was screened using the term
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 0 5 - 0
BBAEXP 91286 18-8-99
384
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
Fig. 1. Proposed pathway of glyoxylate and hydroxypyruvate metabolism in mammalian liver. AGT, alanine:glyoxylate aminotransferase; HPR, hydroxypyruvate reductase; GR, glyoxylate reductase; GO, glycolate oxidase.
`hydroxypyruvate reductase' and a cDNA identi¢ed with peptide sequence homology to HPR from Hyphomicrobium methylovorum. The DNA sequence of this cDNA was used to screen the EST database for clones containing additional 5P-sequence and the longest cDNA clone obtained from the IMAGE Consortium [LLNL] (web site http://bbrp.llnl.gov/bbrp/ image) [8] and sequenced in full. The full-length cDNA clone (IMAGE Consortium clone ID:503200) was ampli¢ed by polymerase chain reaction (PCR) using oligonucleotide primers (Genosys Biotechnologies, Pampisford, UK) designed to introduce restriction sites for KpnI and HindIII at the 5P- and 3P-ends, respectively (forward primer, 5P-ATGGTACCGGGTCGGCGGCTG; reverse primer, 5P-GCAAGCTTCCCTTGGCTCTGC). PCR conditions were as follows: 5 ng DNA, 50 mmol/l potassium chloride, 10 mmol/l Tris HCl, pH 9.0, 0.1% Triton X-100, 2 mmol/l magnesium chloride, 200 Wmol/l dNTP, 0.6 Wmol/l each primer, 0.25 U Taq polymerase (Promega, Southampton, UK) in a volume of 25 Wl. Thirty cycles of 94³C 0.5 min, 66³C 0.5 min, 72³C for 1.5 min were carried out. Following digestion with KpnI and HindIII, the PCR product was ligated into the pTrcHis B expres-
sion vector (Invitrogen, The Netherlands) which had been cut with the same enzymes and the cut ends dephosphorylated. The cDNA was inserted into the multiple cloning site downstream of the His tag and Anti-Xpress antibody epitope and in frame with the plasmid initiation codon. The construct (pTrcHisBHPR) was transfected into Epicurean coli BL21 (DE3) competent cells (Stratagene, Cambridge, UK). Individual colonies were cultured overnight in SOB medium (20 g/l tryptone, 5 g/l yeast extract, 0.5 g/l sodium chloride, 2.5 mmol/l potassium chloride, 10 mmol/l magnesium chloride pH 7.0) containing ampicillin at a ¢nal concentration of 50 Wg/ml. Following plasmid puri¢cation, digestion with KpnI and HindIII con¢rmed the presence of an insert of the correct size (1093 bp). Single colonies of pTrcHisB and pTrcHisB-HPR in BL21 cells were grown up overnight in 2 ml SOB medium plus ampicillin (50 Wg/ml). Aliquots (50 ml) of SOB medium containing ampicillin (50 Wg/ml) were inoculated with 0.2 ml of the overnight cultures and cells grown with aeration to an absorbance at 600 nm of 0.6. Isopropylthio-L-D-galactoside (¢nal concentration 1 mmol/l) was added and the culture incubated for a further 5 h with shaking. The cells were spun down (3000 rpm for
BBAEXP 91286 18-8-99
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
385
Fig. 2. cDNA and predicted protein sequence of human HPR/GR (GLXR). Putative polyA addition signal sequence (AATAA) is underlined. Sequence deposited in GenBank submission no. AF134895.
BBAEXP 91286 18-8-99
386
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
10 min), washed once with PBSA, pelleted again and the pellet sonicated on ice in 1 ml 100 mmol/l potassium phosphate bu¡er, pH 8.0 containing 240 mmol/l sucrose using three, 10-s bursts from a Microson XL sonicator. The cell debris was pelleted by centrifugation at 13 000 rpm for 10 min at 4³C. HPR and GR activity was determined in the supernatant as previously described [6]. Six Wg of total cell protein from sonicate supernatants were electrophoresed in rehydrated polyacrylamide gels (Clean gel 36S, Amersham Pharmacia Biotech, St Albans, UK). After blotting onto nitrocellulose, non-speci¢c binding sites were blocked by immersion in 3% (w/v) milk proteins in PBSA, followed by incubation of the blot overnight with
Anti-Xpress antibody (Invitrogen) at a ¢nal dilution of 1 in 5000. Excess antisera was removed by washing in PBSA (2U10 min) and the blot incubated with alkaline phosphatase conjugated, goat-antimouse IgG (Sigma, Poole) for 3 h at room temperature. After two, 10-min washes in PBSA, the blot was immersed in alkaline phosphatase Colour Development reagent (Bio-Rad, Welwyn Garden City) for 25 min. The cDNA identi¢ed from the IMAGE library is 1235 bp, with 41 bp of 5P-untranslated region and an open reading frame of 987 bp (Fig. 2). The gene encoded by this cDNA has been assigned the symbol GLXR by the Nomenclature Committee of the Human Genome Project. The 3P-untranslated region is
Fig. 3. Peptide sequence of human HPR/GR aligned with glycerate dehydrogenase (NADH-dependent HPR/GR) from Hyphomicrobium methylovorum (hypho) (GenBank P36234), Cucumis sativus (cucurbit) (Genbank P13443) and Methylobacterium extorquens (methylo) (Genbank Q59156).
BBAEXP 91286 18-8-99
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
387
Fig. 4. HPR (shaded boxes) and GR (open boxes) activity in BL21 cells transfected with pTrcHisB-HPR and pTrcHisB, respectively.
225 bp with a putative polyadenylation signal sequence (AATAA) 14 nucleotides from the end of the cDNA. The sequence is predicted to encode a protein of 328 amino acids with a molecular weight of 36.5 kDa (http://www.expasy.ch/tools/pi-tool. html). The protein has sequence similarity with HPR/GR from H. methylovorum (32%), Cucucmis sativus (32%) and Methylobacterium extorquens (21%) (Fig. 3). Expression of the cDNA in BL21 bacterial cells produced a protein with GR and HPR activities of 631 þ 22 and 509 þ 17 nmol NADPH oxidised/min/ mg protein respectively (mean þ 1 S.D. from six analyses) compared with activities of 124 þ 7 nmol/min/ mg protein and 16 þ 8 nmol/min/mg in cells transfected with vector alone (Fig. 4). A fusion protein of 43 kDa was detected on Western blot analysis which was not present in sonicates of cells transfected with vector alone. As the fusion protein adds an additional 3 kDa to the protein size, this result indicates that the protein translated from the cDNA is approximately 40 kDa, slightly bigger than the predicted 36.5 kDa predicted from the amino acid sequence. The same size band was detected under denaturing and non-denaturing conditions suggesting that the enzyme is present as a monomer and is active in this form. In the present study, we have identi¢ed a fulllength cDNA clone from human liver by virtue of its homology to HPR from H. methylovorum. Ex-
pression of the cDNA in BL21 cells produced a fusion protein of 43 kDa with the ability to reduce both glyoxylate and hydroxypyruvate in the presence of NADPH as cofactor. As the plasmid transcript provides an additional 40 amino acids, the actual size of the expressed HPR/GR protein is of the order of 40-kDa. The deduced protein sequence shares approximately 30% sequence similarity with HPR and GR from a number of species of plants and bacteria including H. methylovorum, C. sativus and M. extorquens (Fig. 3). There was no signi¢cant similarity with any of the mammalian lactate dehydrogenase genes. Analysis of the peptide sequence using the Prosite program [9] revealed a 2-hydroxyacid dehydrogenase signature starting at codon 232 (MKETAVFINISRGDVVN). A cDNA of similar size encoding a protein with D-GDH activity has been described in abstract form [10], but no information was given as to its activity with hydroxypyruvate or glyoxylate or its nucleotide or peptide sequence and therefore no further comparisons are possible. However, in view of the fact that GR, HPR and D-GDH activities are all decreased in PH2 [1,4] and therefore that one enzyme appears to have all three activities, it is possible that the two cDNA species are the same. Further studies will elucidate the kinetics of this enzyme, physical properties and role in metabolism. The discovery of this gene also enables the molecular genetics of PH2 to be established.
BBAEXP 91286 18-8-99
388
G. Rumsby, D.P. Cregeen / Biochimica et Biophysica Acta 1446 (1999) 383^388
This project was funded by a grant from the Oxalosis and Hyperoxaluria Foundation. References [1] H. Williams, L.J. Smith, New Engl. J. Med. 278 (1968) 233^ 239. [2] J.E. Willis, H.J. Sallach, J. Biol. Chem. 237 (1962) 910^915. [3] P. Dawkins, F. Dickens, Biochem. J. 94 (1965) 353. [4] J. Mistry, C.J. Danpure, R.A. Chalmers, Biochem. Soc. Trans. 16 (1988) 626^627.
[5] E. Van Schaftingen, J.-P. Draye, F.V. Van Hoof, Eur. J. Biochem. 186 (1989) 355^359. [6] C.F. Gia¢, G. Rumsby, Ann. Clin. Biochem. 35 (1998) 104^ 109. [7] D. Cregeen, G. Rumsby. J. Am. Soc. Nephrol., in press. [8] G.G. Lennon, C. Au¡ray, M. Polymeropoulos, M.B. Soares, Genomics 33 (1996) 151^152. [9] A. Bairoch, P. Bucher, K. Hofmann, Nucleic Acids Res. 25 (1997) 217^221. [10] S.D. Cramer, K. Lin, R.P. Holmes, J. Urol. 159 (5S) (1998) 661.
BBAEXP 91286 18-8-99