Purification and kinetics of reductase from Drosophila melanogaster

Gates, R. L.; Farmer, James L.; Carter, Melvin W.; Bradshaw, William S.

doi:10.1016/0020-1790(83)90062-8

Cited by 3 publications

(1 citation statement)

References 10 publications

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“…This mechanism has also been reported for the P5C reductase purified from Drosophila melanogaster (5), but it has not previously been reported for plants. Such substrate inhibition is unlikely to be important in vivo, where the concentration of P5C and NADH are expected to occur at less than millimolar concentrations but has to be kept in mind when assaying P5C reductase in vitro.…”

Section: Pyndine Nucleotide Specificitymentioning

confidence: 79%

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Laliberté¹,

Hellebust²

1989

Plant Physiol.

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Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCI buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCI, KCI, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.A-Pyrroline-5-carboxylate reductase (L-Proline:NAD(P)-5-oxidoreductase, EC 1.5.1.2) catalyzes the final step in the biosynthetic pathway leading from glutamic acid to proline. The enzyme has been partially purified and characterized for many higher plants (7,9,13,17,21,23). None ofthese studies suggest a major role for P5C2 reductase in the regulation of proline biosynthesis. However, the activity of P5C reductase has been shown to increase 3.5-fold when seedlings of Pennisetum typhoides were grown in the presence of 17 mm NaCl with a concomitant increase in proline concentration (7). More recently, Treichel (23) (15). In these cells, glucose-6-phosphate dehydrogenase, which catalyzes the rate-limiting step of the pentose phosphate pathway, is dependent on the availability of NADP+ and is inhibited by NADPH. It is believed that P5C reductase by reducing P5C to proline with NADPH as cofactor serves to furnish NADP+ to glucose-6-phosphate dehydrogenase for the synthesis of purine nucleotides. Recently, it has been shown that in soybean nodules P5C reductase can indeed play an important role for the support of purine biosynthesis in addition to producing proline for incorporation into protein (8). In both erythrocytes and soybean nodules the P5C reductases are characterized by a higher affinity for NADPH than NADH, and a sensitivity to inhibition by NADP+ but not by proline. Finally, as pointed out by Bellinger and Larher (2), in glycophytes and in some halophyt...

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Section: Pyndine Nucleotide Specificitymentioning

confidence: 79%

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Laliberté¹,

Hellebust²

1989

Plant Physiol.

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Small Nitrogenous Compounds

Urich

1994

Comparative Animal Biochemistry

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Misener

Walker

2001

Biochemical Genetics

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The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster. The cDNA sequence was identified by interspecies complementation of an E. coli proline auxotroph and encodes a protein 280 amino acids in length with 25-41% identity to pyrroline 5-carboxylate reductases isolated from other organisms. The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region. With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy. The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer. Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism.

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Purification and kinetics of reductase from Drosophila melanogaster

Cited by 3 publications

References 10 publications

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Small Nitrogenous Compounds

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