Joannès Nari

The formation and reactivity of peroxidase Compound III

Abstract 1. 1. Indoleacetic acid can, under certain conditions (acidic medium in particular), reduce horseradish peroxidase (donor:H2O2-oxidoreductase, EC 1.11.1.7). It can also reduce phenosafranine, the oxidation-reduction potentials of which, at various pH values, are very close to those of peroxidase. These results are obtained in the absence of peroxidase in the medium. They confirm those described in an earlier report. 2. 2. If ferriperoxidase is reduced by H2 or by the semiquinone of methylviologen, the appearance of the Compound III spectrum may be recorded in the presence of O2. Compound III obtained under these conditions is therefore identifiable with oxyferroperoxidase. 3. 3. Oxyferroperoxidase appears to be capable of oxidizing indoleacetic acid and catechol. This last substance can also be destroyed by the ferriperoxidase-indoleacetate system. Catechol degradation is achieved under these conditions by 2 different mechanisms: 1. (a) A peroxidation reaction leading, probably, to the appearance of o- benzoquinone ; 2. (b) A degradation reaction of a different type (perhaps an oxygenation) conditioned by the ferroperoxidase-oxyferroperoxidase system, insensitive to cyanide, and leading to the appearance of substances different from o- benzoquinone .

Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds

Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.

Contribution a l’etude des mecanismes de la degradation de l’acide β-indolylacetique par la peroxydase de raifort

Summary 1. Indoleacetic acid degradation catalyzed by horseradish peroxidase (donor: hydrogen-peroxyde oxidoreductase, EC 1.11.1.7), in the absence of peroxide introduced into the medium, may be inhibited by carbon monoxide. This inhibition is partially reversed by light. Indoleacetic acid, in the absence of free oxygen, and in the absence or presence of peroxide, fails to appreciably reduce the peroxidase, On the other hand, if the medium contains any CO, carboxyferroperoxidase very quickly appears. If one decomposes this carboxyferroperoxidase with light, it does not form ferroperoxidase but ferriperoxidase. These results suggest the existence of the reaction:ferriperoxidase + indoleacetic acid ⇌ ferroperoxidase + indoleacetic acid’This equilibrium would normally be moved towards the left. The combination of ferroperoxidase with CO, however, allows the reduction of appreciable quantities of enzyme. 2. This ferroperoxidase effectively participates, under certain conditions, in indoleacetic acid degradation. 3. Indoleacetic acid destruction can be achieved by two different mechanisms: (a) a typical peroxidation reaction bringing into play ferriperoxidase, compound II and indoleacetic acid free radicals; (b) a degradation cycle complicating the intervention of ferroperoxidase and of compound III. This cycle is only functional in acidic medium (pH 3–4). The first system is insensitive to CO and ferricyanide, whereas the functioning of the second is blocked by these substances.

Purification and molecular properties of an acid phosphatase from Asclepias curassavica latex

Abstract An acid phosphatase was isolated and purified from Asclepias curassavica latex. This phosphatase is the first to be obtained in the pure state from non-articulated laticifers. The enzyme has a molecular weight close to 27 000 and its amino acid composition was determined. It catalyses the hydrolysis of various phosphorylated compounds, p- nitrophenyl phosphate being the most efficient as found for most other phosphatases. The optimal pH was found to be approx. 6.0 and at this pH the Michaelis constant was 0.69 mM. Phosphate was a competitive inhibitor indicating that it is the last product to be liberated during the reaction course. Pyrophosphate, arsenate, molybdate, cupric and mercuric salts were inhibitory. Calcium, magnesium, potassium and tungsten had no effect. The physiological significance of the presence of this enzyme in the latex was discussed.