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The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.

Peroxisomes from spinach leaves, mungbean hypocotyls, and potato tubers catalyze a palmitoyl-CoA-dependent, KCN-insensitive O2 uptake. In the course of this reaction O2 is reduced to H2O2 in a 1:1 stoichiometry and palmitoyl-CoA oxidized, in a 1:1 stoichiometry, to a product serving as substrate for enoyl-CoA hydratase. These findings demonstrate the existence of a peroxisomal acyl-CoA oxidase in these tissues. Enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) are also associated with the peroxisomes from mung-bean hypocotyls and potato tubers (as well as with spinach leaf peroxisomes as recently reported; Gerhardt 1981, FEBS Lett. 126, 71). The low activities of these enzymes in mitochondrial fractions seem to be due to contaminating peroxisomes since the ratio of β-oxidation enzyme activities to catalase activity did not significantly differ between peroxisomal and mitochondrial fractions isolated on sucrose density gradients. The proof of localization of β-oxidation enzymes in peroxisomes without glyoxysomal function leads to the concept that fatty-acid oxidation is a consistent basic function of the peroxisome in cells of higher plants.

Branched-chain 2-oxo acids which are formed by transamination of leucine, isoleucine, and valine are metabolized by the peroxisomes from mung bean (Vigna radiata L.) hypocotyls. Acylcoenzyme A (CoA) thio ester intermediates of the pathways were separated by reversed-phase high performance liquid chromatography. Retention time and cochromatography of individual acyl-CoA reference standards were used for identification of the acyl-CoA esters separated from the assay mixtures. Based on the results of identification and those of kinetic experiments, pathways of the peroxisomal degradation of 2-oxoisocaproate, 2-oxoisovalerate, and 2-oxo-3-methylvalerate are suggested.Peroxisomes are common organelles of higher plant cells. A basic metabolic function of these organelles seems to be fatty acid degradation (7). Peroxisomes degrade by ,8-oxidation saturated straight chain, long-, medium-and short-chain fatty acids (8). Unsaturated fatty acids appear also to be degraded by peroxisomes. The enzymes required to link the catabolism of unsaturated fatty acids to the fl-oxidation sequence have recently been demonstrated in glyoxysomes, the peroxisomes of lipid-storing nutrient tissues of seeds (2).Glyoxysomes are involved in the conversion of reserve lipid to sucrose during germination (1). The physiological role of the peroxisomal fatty acid degrading system in non-lipid storing tissues, i.e. in the majority of plant tissues, has yet to be elucidated. The turnover of membrane lipids has to be considered as a source of fatty acids in non-lipid storing tissues. Unsaturated fatty acids would be the main substrates for the peroxisomal fl-oxidation system. The data on the ability of glyoxysomes to degrade unsaturated fatty acids (2) support this concept.A second physiologically important source of substrate for a fatty acid degrading system in non-lipid storing tissues can result from the degradation of branched-chain amino acids in the course of steady-state protein turnover. In Lemna minor, 50 to 60% of the leucine and isoleucine resulting from protein turnover is metabolized (3). Intermediates of the catabolism of leucine, isoleucine, and valine are branched-chain 2-oxo acids. The catabolism of these acids in higher plants has received very little attention up to now ( 12). We have recently shown that the peroxisomes but not the mitochondria from a non-lipid storing tissue are able to activate by oxidative de-' Dedicated to Professor Achim Trebst on the occasion of his 60th birthday.

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