Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.
Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.
Turner
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SR
Cambridge Laboratory, AFRC Institute of Plant Science Research, John Innes Centre, Norwich, United Kingdom.
Ireland
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R
Morgan
C
C
Rawsthorne
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S
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics*; D007887:Fabaceae / Q000201:enzymology* / Q000648:ultrastructure; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010946:Plants, Medicinal; D016415:Sequence Alignment
D007527:Isoenzymes; D004247:DNA; D012696:Glycine Hydroxymethyltransferase