Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.).
Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) has been purified from potato tuber at least 850-fold to apparent homogeneity as judged by SDS-PAGE. The enzyme was characterized by Km values of 260 microM for glucose-6-phosphate and 6 microM for NADP and a broad pH optimum between pH 7.5 and 9. NADPH, GTP, ATP, acetyl CoA and CoA inhibited G6PDH activity. Dithiothreitol (DTT) did not inactivate the enzyme. A highly specific antiserum was produced in a rabbit and used for immunodetection of G6PDH in Western blots. A cDNA library from potato leaves was screened with DNA probes produced by the polymerase chain reaction (PCR) in the presence of g6pdh-specific primers. A full-length cDNA clone was analyzed and the derived amino acid sequence compared with known G6PDH sequences from various sources. The homology of the plant sequence with G6PDH sequences from animals and yeast was found to be rather high (52%), whereas there was significantly lower homology with sequences of bacterial origin (37%). The lack of a plastidic signal sequence as well as the insensitivity of the recombinant enzyme towards reduced DTT, support the view that the cDNA sequence of a redox-independent cytosolic isoform was obtained.
Graeve
K
K
Pflanzenphysiologie, FB 5 Biologie/Chemie, Universität Osnabrück, Germany.
von Schaewen
A
A
Scheibe
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*
D000596:Amino Acids; D018076:DNA, Complementary; D007527:Isoenzymes; D005954:Glucosephosphate Dehydrogenase