Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1-(14)C]C24:0 for peroxisomal beta-oxidation to generate [1-(14)C]acetyl-CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2-(14)C]acetate and [1-(14)C]C8:0 but not from [1-(14)C]C24:0, implicating a peroxisomal, lovastatin-resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1-(14)C]C24:0-derived [1-(14)C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS.     

Association of monoamine oxidase and malate dehydrogenase with liver peroxisomes of genetically obese (ob/ob and db/db) mice.

1. Liver post-nuclear supernatants (PNS) from genetically obese (ob/ob and db/db), lean (+/?), and albino mice were fractionated by dual centrifugation in B-XIV zonal rotors and subcellular fractions were analysed by marker-enzyme estimations and by electron microscopy. 2. Rate-dependent banding of PNS yielded a peroxisome-enriched region (PER) well-separated from mitochondria. 3. Density-dependent banding of PER in ob/ob and db/db mice only, yielded purified peroxisomes which were associated with malate dehydrogenase (cytosolic) and monoamine oxidase. 4. Markers for the mitochondrial matrix, intermembrane space and inner membrane compartments were absent from the peroxisomes. 5. The experimental results are interpreted as indicating that peroxisomes of genetically obese mice are either altered so that protein import is imprecise or so that their attachment to mitochondria is more extensive.     

Effect of acidotropic amines on the accumulation of newly synthesized membrane and luminal proteins in Chinese-hamster ovary (CHO) cell lysosomes.

Lysosomes were isolated by sequential gradient centrifugation [Madden, Wirt & Storrie (1987) Arch. Biochem. Biophys. 257, 27-38] from control or acidotropic-amine-treated Chinese-hamster ovary (CHO) cells. By marker-enzyme analysis, the preparation from chloroquine or NH4Cl-treated cells was about 25-fold enriched for lysosomes compared with the postnuclear supernatant and contained little or no marker activities for the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, mitochondria, cytosol and peroxisomes. The yield of amine-treated lysosomes was about 60% relative to the postnuclear supernatant. Electron microscopy and cytochemistry demonstrated that the amine-treated preparation was highly purified. Cytochemical analyses after a short-term pulse of horseradish peroxidase revealed that endosomal contamination of the lysosomal preparation was less than 1%. Lysosomal polypeptides were biosynthetically labelled with [35S]methionine and identified by SDS/polyacrylamide-gel electrophoresis. As expected, the bulk accumulation of luminal proteins into lysosomes was decreased. The bulk accumulation of membrane proteins was increased by acidotropic amine treatment. There were also several qualitative differences in each lysosomal compartment, with new species observed and other species absent. These data suggest that a low pH is not necessary for the normal accumulation of the bulk of membrane proteins in lysosomes and that membrane trafficking from Golgi apparatus to lysosomes occurs at a high rate in acidotropic-amine-treated CHO cells.     

Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the 13C labeling of malonyl-CoA and the acetyl moiety of citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.     

Subcellular fractionation evidence for a putative peroxisome-mitochondrion attachment in the liver of normal and genetically obese (ob/ob and db/db) mice.

1. Liver post-nuclear supernatants (PNS) from several mouse strains were fractionated by zonal centrifugation and fractions analysed by marker-enzyme estimations+electron microscopy. 2. Rate-dependent banding of PNS yielded peroxisome-enriched (PER) and mitochondrion-enriched (MER) regions. 3. Density-dependent banding of PER yielded peroxisomes (approximately 1.22 g/ml) well separated from mitochondria (approximately 1.8 g/ml). 4. Density-dependent banding of MER yielded peroxisomes that co-distributed with mitochondria and electron microscopy revealed close proximity of the two organelles. 5. Experiments demonstrated that co-distribution was not due to weak binding of proteins or to agglutination of organelles. 6. The results indicate in vivo attachment of some mitochondria and peroxisomes.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

The Zellweger syndrome: deficient chain-shortening of erucic acid (22:1 (n-9)) and adrenic acid (22:4 (n-6)) in cultured skin fibroblasts

In the Zellweger syndrome where peroxisomes are absent, extremely long fatty acids (24:0 and 26:0) accumulate in tissues suggesting that these fatty acids are normally beta-oxidized in the peroxisomes. Previous studies with rat hepatocytes suggest that peroxisomes are also important in oxidation of C22 unsaturated fatty acids. This study shows that cultured fibroblasts from normal human controls shorten [14-14C]erucic acid (22:1(n-9)) to oleic acid (18:1(n-9)) efficiently while Zellweger fibroblasts are deficient in chain-shortening. [2-14C]Adrenic acid (22:4(n-6)) is oxidized in control fibroblasts probably by chain-shortening to arachidonic acid (20:4(n-6)). Only a little adrenic acid is oxidized in Zellweger fibroblasts. Linolenic acid (18:3(n-3)) is desaturated and chain-elongated in both control and Zellweger fibroblasts. The results support the view that peroxisomes play a normal physiological role in the shortening of C22 unsaturated fatty acids and that this function is deficient in Zellweger fibroblasts.     

Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds

[3H]nafenopin, a known inducer of liver peroxisomal enzymes, was shown to bind to a specific, saturable pool of binding sites in cytosols from rat liver and kidney cortex. Tissue levels of this binding protein (liver greater than kidney cortex; not detectable in myocardium, skeletal muscle) were seen to correlate with the ability of nafenopin to induce peroxisomal enzymes in these organs. Clofibrate and ciprofibrate, which are structurally similar to nafenopin, competitively blocked the specific binding of [3H]nafenopin. Phenobarbital, a non-inducer of peroxisomes, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamide, which are structurally unrelated peroxisome proliferators, did not complete for the specific [3H]nafenopin binding sites. The [3H]nafenopin binding protein is proposed as a mediator of the drug-induced increase in peroxisomes and associated peroxisomal enzymes.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Metabolism of dicarboxylic acids in rat hepatocytes

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.     

Metabolism of acetyl-CoA by isolated peroxisomal fractions: formation of acetate and acetoacetyl-CoA.

Liver peroxisomal fractions, isolated from rats treated with clofibrate, were shown to hydrolyze added [1-14C]acetyl-CoA to free [1-14C]acetate. [1-14C]Acetyl-CoA was, however, also converted to [14C]acetoacetyl-CoA. This reaction was inhibited by added ATP and by solubilization of the peroxisomes. The effect of ATP on synthesis of [14C]acetoacetyl-CoA was likely due to ATP-dependent stimulation of acetyl-CoA hydrolase (EC 3.1.2.1) activity. The inhibitory effect due to solubilizing conditions of incubation remains unexplained. During peroxisomal beta-oxidation of [1-14C]palmitoyl-CoA, [1-14C]acetyl-CoA, [1-14C]acetate, and [14C]acetoacetyl-CoA were shown to be produced. Possible metabolic implications of peroxisomal acetoacetyl-CoA synthesis are discussed.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.     

Degradation of very long chain dicarboxylic polyunsaturated fatty acids in mouse hepatocytes, a peroxisomal process

Polyunsaturated fatty acids can be omega-oxidized to dicarboxylic polyunsaturated fatty acids (DC-PUFA), bioactive compounds which cause vasodilatation and activation of PPARalpha and gamma. DC-PUFA can be shortened by beta-oxidation, and to determine whether mitochondria and/or peroxisomes are responsible for this degradation 20-carboxy-[1-(14)C]-eicosatetraenoic acid (20-COOH-AA) was synthesized and given to hepatocytes from mouse models with peroxisomal dysfunctions. In contrast to wild type cells, hepatocytes from mice with liver-selective elimination of peroxisomes, due to Pex5p deficiency, failed to produce (14)CO(2) and labeled acid-soluble oxidation products, indicating that peroxisomes are involved in the degradation of 20-COOH-AA. Subsequently, the oxidation of 20-COOH-AA was analyzed in hepatocytes lacking multifunctional protein 1 (MFP1) or MFP2, key enzymes of the peroxisomal beta-oxidation. Degradation of 20-COOH-AA was partially impaired in MFP1, but not in MFP2 knockout hepatocytes. Taken together, peroxisomes and not mitochondria are the site of beta-oxidation of DC-PUFA, and MFP1 is involved in this process.     

Hepatic oxalate production: the role of hydroxypyruvate

The metabolism of hydroxypyruvate to oxalate was studied in isolated rat hepatocytes. [14C]Oxalate was produced from [2-14C]- and [3-14C]- but not [1-14C]hydroxypyruvate. No oxalate was produced from similarly labeled pyruvate. The mechanism by which hydroxypyruvate is metabolized to oxalate involves decarboxylation at the carbon 1 position as the initial step. This activity was distinct from that which produced CO2 from the carbon 1 position of pyruvate. Hydroxypyruvate decarboxylase activity was found mainly in the mitochondria, with the remainder (25%) in the cytosol. No activity was present in the peroxisomes, the probable site of oxalate production from glycolate and glyoxylate. Hydroxypyruvate, but not pyruvate stimulated [14C]oxalate production from [U-14C]fructose, suggesting that hydroxypyruvate is either an intermediate in the fructose-oxalate pathway, or that it prevents carbon from leaving that pathway. The lack of effect of pyruvate in this regard is evidence against redox being the primary effect of hydroxypyruvate and focuses attention on hydroxypyruvate and its precursors as important sources of carbon for oxalate synthesis from both carbohydrate and protein.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

ATP binding/hydrolysis by and phosphorylation of peroxisomal ATP-binding cassette proteins PMP70 (ABCD3) and adrenoleukodystrophy protein (ABCD1)

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP when peroxisomes were incubated with these nucleotides at 37 degrees C in the absence Mg2+ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg2+ reduced the photoaffinity labeling of PMP70 with 8-azido-[gamma-32P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[alpha-32P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg2+, the bound ATP is hydrolyzed to ADP in the presence of Mg2+, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

Biosynthesis of dolichol by rat liver peroxisomes

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl-P2 or [3H]farnesyl-P2 in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di-2-ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Glyoxylate decarboxylation during photorespiration

At 25° C under aerobic conditions with or without gluamate 10% of the [1-¹⁴C]glycollate oxidised in spinach leaf peroxisomes was released as ¹⁴CO₂. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO₂ release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce ¹⁴CO₂ release from either [1-¹⁴C]glycollate or [1-¹⁴C]glyoxylate. Furthermore, the amount of available H₂O₂ (Grodzinski and Butt, 1976) was sufficient to account for all of the CO₂ release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure available H₂O₂ directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO₂ release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H₂O₂ at a known rate was used to replace the chloroplasts, similar rates of ¹⁴CO₂ release and [¹⁴C]glycine synthesis from [1-¹⁴C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO₂ arises during glyoxylate decarboxylation under the action of H₂O₂. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - INH isonicotinylhydrazide - PHMS pyridyl-2-yl--hydroxymethane sulphonic acid