Purification and Characterization of Cytoplasmic NADP+-Isocitrate Dehydrogenase, and Amplification of the Nadp+-IDH Gene from the Wing-Dimorphic Sand Field Cricket, Gryllus firmus

摘要

Cytoplasmic NADP+-isocitrate dehydrogenase (NADP+-IDH) has been purified and characterized, and its gene sequenced in many animal, plant, and yeast species. However, much less information is available on this enzyme-gene in insects. As a first step in investigating the biochemical and molecular mechanisms by which NADP+-IDH contributes to adaptations for flight vs. reproduction in insects, the enzyme was purified to homogeneity in the wing-dimorphic cricket, Gryllus firmus, characterized, and its corresponding gene sequenced. Using a combination of polyethylene glycol precipitation, Cibacron-Blue affinity chromatography, and hydrophobic interaction chromatography the enzyme was purified 291-fold (7% yield; specific activity = 15.8 µmol NADPH/min/mg protein). The purified enzyme exhibited a single band on SDS PAGE (46.3 kD), but consisted of two N-terminal amino acid sequences that differed in the first two amino acids. Purified enzyme exhibited standard Michaelis-Menten kinetics at pH 8.0 and 28° C (KM(NADP+) = 2.3 ± 0.4 µM; KM(Na+-Isocitrate) = 14.7 + 1.8 µM). Subunit molecular mass and KMS were similar to published values for NADP+-IDHs from a variety of vertebrate and two insect species. PCR amplification of an internal sequence using genomic DNA followed by 3′ and 5′ RACE yielded a nucleotide sequence of the mature protein and translated amino-acid sequences that exhibited high similarity (40–50% and 70–80%, respectively) to sequences from insect and vertebrate NADP+-IDHs. Two potential ATG start codons were identified. Both Nterminal amino-acid sequences matched the nucleotide sequence, consistent with both enzyme forms being transcribed from the same gene, although these variants could also be encoded by different genes. Bioinformatic analyses and differential centrifugation indicated that the majority, if not all, of the enzyme is cytoplasmic. The enzyme exhibited high specific activity in fat body, head and gut, and a single band on native PAGE.