SummaryIn a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sun¯ower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate speci®city and with hydrogen peroxide (H 2 O 2 ) as one of the reaction products. The sun¯ower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.

The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.

We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.

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e have investigated the palmitic acid (cl6: o) and cerotic acid (c26: o) activating activities in rat-her microsomes and peroxisomes. The activation of the two fatty acids showed similar dependencies on ATP and coenzyme A, reflected in about equal apparent K, values both in microsomes and peroxisomes. In microsomes and peroxisomes similar apparent K, values for palmitic acid were found (15 pM and 22.8 pM, respectively), whereas apparent K, values for cerotic acid were 8.4 pM and 1 .O pM in microsomes and peroxisomes, respectively. The activation of cerotic acid was found to be inhibited to a progressively greater extent by increasing concentrations of 1-pyrenedecanoic acid (P10) as compared to the activation of palmitic acid, both in microsomes and peroxisomes. The inhibition by P10 of palmitic acid activation and cerotic acid activation was non-competitive in both organelles. From the observation that PI0 activation is not affected by palmitic acid and cerotic acid, we conclude that PI0 is activated by a distinct enzyme. Furthermore, our results are in accordance with earlier suggestions that activation of cerotic acid is brought about by an enzyme distinct from the palmitoyl-CoA synthetase.Mammalian cells contain a number of different fatty acid :CoA ligases (alternatively called acyl-CoA synthetases) capable of activating a variety of fatty acids to their corresponding acyl-coenzyme A esters (see [l] for review). Despite intensive research over the last few years there are differences of opinion about many aspects of fatty acid activation. With regard to the activation of long-chain fatty acids, it is clear that mitochondria, endoplasmic reticulum and peroxisomes contain ATP-dependent long-chain-acyl-CoA synthetase activity [2 -41. This activity is membrane-bound in all three organelles and the active sites of the enzymes face the cytosol in the case of the endoplasmic reticulum and peroxisomes [4, 51. The mitochondria1 long-chain-acyl-CoA synthetase is an integral part of the outer membrane, although its exact orientation in the membrane is disputed [6, 71. Tanaka and coworkers [8] purified the long-chain-acyl-CoA synthetase from mitochondria and microsomes of rat liver and concluded that the enzyme proteins are indistinguishable as judged by physicochemical, catalytic and immunological properties. Elaborating on this study, Miyazawa et al. [9] concluded that the peroxisomal enzyme as well is identical to the other two