We have previously identified and purified a novel β-glucosidase, designated PNGH (pyridoxine-5´-β-d-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5´-β-d-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase–phlorizin hydrolase), the β-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568–784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.
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Research Article|
May 15 2004
Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene
Chi-Wah TSEUNG;
Chi-Wah TSEUNG
*Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611-0370, U.S.A.
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Laura G. McMAHON;
Laura G. McMAHON
*Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611-0370, U.S.A.
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Jorge VÁZQUEZ;
Jorge VÁZQUEZ
†Physiology and Functional Genomics Department, College of Medicine, University of Florida, Gainesville, FL 32610, U.S.A.
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Jan POHL;
Jan POHL
‡Microchemical Facility, Emory University School of Medicine, Atlanta, GA 30322, U.S.A.
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Jesse F. GREGORY, III
Jesse F. GREGORY, III
1
*Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611-0370, U.S.A.
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
September 16 2003
Revision Received:
February 18 2004
Accepted:
February 19 2004
Accepted Manuscript online:
February 19 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (1): 211–218.
Article history
Received:
September 16 2003
Revision Received:
February 18 2004
Accepted:
February 19 2004
Accepted Manuscript online:
February 19 2004
Citation
Chi-Wah TSEUNG, Laura G. McMAHON, Jorge VÁZQUEZ, Jan POHL, Jesse F. GREGORY; Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene. Biochem J 15 May 2004; 380 (1): 211–218. doi: https://doi.org/10.1042/bj20031416
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