The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergendphospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate V , and the kinetic constant K:, were determined with the micellar substrate.The specificity of various substrates was examined by comparing the V,,,IK,B, values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.Keywords: phospholipase D ; phospholipid; mixed micelles; sunflower oil bodies.Phospholipase D (PLD) catalyses the hydrolysis of the ester linkage between phosphatidic acid and various alcohol moieties of several phospholipid species [I-31. This enzyme is widespread in the plant and animal kingdoms. It has been reported to play an important role in signal-transduction cascades in a variety of mammalian cells [4], where it appears to be activated by various hormones and neurotransmitters and some growth factors. In plants PLD is known to be involved in several cellular processes but its role has not been established clearly. The enzyme is present in the protein bodies of germinating mung bean cotyledons [5] and is associated with plasma and intracellular membranes in seedling tissues of castor bean [6]. Activity changes have been observed in conjunction with water stress [7], senescence [S] and pathogenic infection [9]. During mung bean seed germination and seedling growth, the decrease in the phospholipid content has been correlated with an increase in PLD activity [S]. In castor bean endosperm, immunoblot analyses have shown that the amount of PLD increases during the first five days of germination. It has therefore been suggested that these activity changes may be due to metabolic reactions involving membrane phospholipids, which are essential to plant growth and development [lo]. In rice bran, crude PLD prepara- (EC 3.1.4.4). tions were found to be able to react ...
Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol. Its specificity towards sedoheptulose 1,7-bisphosphate versus fructose 1,6-bisphosphate is high, but not absolute.
This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-l,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg"] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed V,,, which is 60% of the theoretical V,,, while that of the oxidized form is only 37% of the theoretical V,,,. The reduced enzyme appears more efficient than the oxidized one in catalysis.
This paper compares structural, immunological and kinetic properties of corn (C,) and spinach (C,) NADPmalate dehydrogenases. These chloroplastic enzymes are regulated in vivo by thiol -disulfide interchange. Both in their oxidized (inactive) and reduced (active) states these enzymes have a dimeric structure with molecular masses for the subunit ranging from 28 kDa to 38 kDa according to the procedure used for the determination. These enzymes are thus structurally related. The use of specific antibodies showed that they are also immunologically related although not identical. Finally both enzymes showed close kinetic properties with comparable k,,, and K,. Since C, plants have approximately ten times more NADP-malate dehydrogenase activity than C3 plants, these data suggest that the differences in activities are probably related to the enzyme content of each plant type.N ADH-dependent malate dehydrogenases are ubiquitous enzymes, isoenzymes of which are found in bacteria, mammals and plants [l]. The latter organisms, however, possess a unique NADPH-dependent malate dehydrogenase (NADP-MDH). The most striking difference between the NADH and the NADPH-dependent malate dehydrogenases is that the latter, unlike its NADH counterparts, is regulated by its redox status [2, 31. Indeed, the NADP-malate dehydrogenase is in vivo activated by light and inactivated in the dark [4, 51. Studies of the activation/inactivation mechanism have led to the discovery of a light-dependent modification system, endogeneous to the chloroplast, called the ferredoxin-thioredoxin system, which can be reconstituted in vitro and comprises isolated thylakoids and the following soluble proteins: ferredoxin, ferredoxin-thioredoxin reductase and thioredoxins [6, 71. Photochemically or chemically (dithiothreitol) reduced thioredoxins can, in turn, activate the NADP-malate dehydrogenase presumably via a thiol -disulfide interchange mechanism with the appearance of new thiol groups on the enzyme together with the catalytic activity [8, 91. NADP-MDH can also be reduced by dithiothreitol alone with a lower efficiency through a similar mechanism [lo-121.NADP-malate dehydrogenase is present only in the chloroplasts of C3 and C4 mesophyll plant cells [13,14]. While it seems to be clear that in C4 leaves it is involved in the soCorrespvndence to J
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