Nicotinamide Adenine Dinucleotide-dependent Proline Dehydrogenase in Chlorella

Abstract

An NAD-linked dehydrogenase from Chlorella pyrenoidosa Chick catalyzing the conversion of L-proline to A1-pyrroline-5-carboxylic acid was partially purified. A.-Pyrroline-5-carboxylic acid was identified as the product by co-chromatography of it and its o-aminobenzaldehyde derivative with authentic compounds. The enzyme is NAD and L-proline specific and is not an oxidase; NADP is not inhibitory. The Michaelis constant for NAD is 0.08 mM and for proline is 0.73 mM.Plants catabolize proline to glutamate (1,5,14,17), and recently it has been shown that extensive catabolism of proline can occur in leaves low in carbohydrate and high in proline (17) and in Chlorella (14). Labeled A&-pyrroline-5-carboxylic acid has been recovered from plant extracts after exposure of the tissue to 14C-proline (5). In animals and microorganisms, the first enzyme in the proline to glutamate conversion is proline oxidase (2,6,7,9). This enzyme requires molecular oxygen, is particulate, is inhibited by cyanide, and cytochrome c will serve as the electron acceptor. The product of the reaction is P5C.2 A similar enzyme has not been isolated from plant tissue. Recently, a proline dehydrogenase has been isolated from peanut (13) and there are preliminary reports that proline dehydrogenase can be isolated from wheat germ (12) and pumpkin cotyledons (16).This paper reports the presence of a proline dehydrogenase in extracts from Chlorella cells. It has been reported that proline dehydrogenase from peanut (13) does not produce P5C; however, we present evidence that P5C is the product of proline dehydrogenase from Chlorella pyrenoidosa. Molecular oxygen is not required for the production of PSC. yellow complex produced by reaction of P5C with oAB; P2C-oAB: yellow complex produced by reaction of P2C with oAB; DEAE: diethylaminoethyl. MATERIALS AND METHODSAfter washing twice, the cells were resuspended in 0.1 time the original volume of 50 mm phosphate buffer (pH 7.2) containing 1 mM Na2EDTA and 1 mm dithiothreitol. The material was passed twice through a French pressure cell at 20,000 p.s.i. Subsequently, the extracts were kept chilled on ice or the operations were done in a coldroom. After centrifugation at 1 OOOg for 10 min, solid (NH4)2SO4 was added to the supernatant at the rate of 176 g/l. After centrifugation at 10,000g for 10 min, the new supernatant was treated with 273 g of (NH4),SO4/ 1. After centrifugation, the pellet was dissolved in the homogenizing buffer.Some purification was obtained by passing the extract through a DEAE-cellulose column (2.1 X 30 cm). A linear gradient of 100 ml of 0.05 M phosphate (pH 7.2) to 100 ml of 0.2 M phosphate (pH 7.2) was used to elute the enzyme. The enzyme solution was dialyzed against a saturated solution of (NH4),S04 (4 C) for 12 hr. Storage was either in saturated (NH4)2SO4 or by freezing after removal of the (NH4)2SO4.Assay for Proline Dehydrogenase. A typical reaction solution contained initial concentrations of 0.17 M Na-carbonate buffer (pH 10.2), 0.48 to 1.9 mm NAD, 6.7 mm proline, and ...

Oxidation of Proline by Plant Mitochondria

“…On the other hand, all evidence presented here is consistent with the operation of a mitochond;ial proline oxidase, perhaps similar to the enzyme reported in rat liver (7,21). Our findings do not negate the possibility that proline dehydrogenase may catalyze proline metabolism in other tissues (10)(11)(12)19), or in the cytoplasm of the species examined in this study.…”

“…Activity of this enzyme apparently has not been demonstrated in higher plants. Instead, several workers have reported the presence of proline dehydrogenase, a nonparticulate, NADlinked enzyme which has been suggested, but not proved, to catalyze P5C formation in vivo (10)(11)(12)19). Doubt that proline dehydrogenase is the in vivo catalyst for proline oxidation has arisen from the necessity to assay it at high pH (above pH 10, refs.…”

“…Doubt that proline dehydrogenase is the in vivo catalyst for proline oxidation has arisen from the necessity to assay it at high pH (above pH 10, refs. 10 and 12) and from the observation that it co-purifies with P5C reductase (10), the NADH-linked proline biosynthetic enzyme that is typically stable and present in relatively high activity in a variety of tissues (6,10,12,16,17). These considerations led us to look for proline oxidation by mitochondria isolated from higher plants.…”

Proline Oxidase and Water Stress-induced Proline Accumulation in Spinach Leaves

“…After the reaction, an additional pink spot was observed further away from the origin than the proline spot; it was identified as P5C. In the present solvent system of paper chromatography, P2C migrates slower than proline and P5C (16). We conclude that P5C was the reaction product that reacted quantitatively with o-aminobenzaldehyde (Fig.…”

“…Synthetic PSC was also spotted separately with and without proline for reference. Descending chromatography was run in 1-butanol-acetic acid-water (4:1:5) (16). After 24 h, the chromatographic paper was dried and sprayed with 0.2% ninhydrin in acetone.…”