Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is unable to grow on methanol because it lacks the enzymes required for its metabolism. To study the possibility of whether or not the methanol oxidation pathway of Hansenula polymorpha can be transferred to S. cerevisiae, the gene coding for alcohol oxidase, a peroxisomal homo-octameric flavoprotein, was introduced into S. cerevisiae. Transformed cells contain varying amounts of alcohol oxidase depending on the plasmid used. Immunocytochemical experiments indicate that the protein is imported into peroxisomes, whether organelle proliferation is induced or not. Cells lack alcohol oxidase activity however, and only the monomeric, non-functional, form of the protein is found. These findings indicate that the H. polymorpha peroxisomal targeting signal of alcohol oxidase is recognized in S. cerevisiae and protein monomers are imported.     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes.

Acyl-CoA oxidase is a major induced enzyme in peroxisomes of Candida tropicalis grown on fatty acids. The gene, POX4, encoding acyl-CoA oxidase was expressed in vitro, and the resulting polypeptide was imported into purified peroxisomes in a temperature-dependent fashion. Plasmids containing fragments of POX4 were prepared, expressed and the polypeptides tested for import into peroxisomes. We identified two regions of acyl-CoA oxidase (amino acids 1-118 and 309-427) that contained information that specifically targeted fragments of acyl-CoA oxidase to peroxisomes. The corresponding regions of the gene were fused to cDNA encoding the cytosolic enzyme dihydrofolate reductase (DHFR), and the expressed fusion proteins were likewise imported into peroxisomes. DHFR itself neither bound to, nor was imported into peroxisomes. Thus, there are at least two regions of peroxisomal targeting information in the acyl-CoA oxidase gene.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.     

Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes

The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.     

The carboxy-terminal region of SMAP2 directs subcellular localization as well as Arf protein specificity

Small G proteins play a central role in the organization of secretory and endocytotic pathways. The recruitment of some effectors, including vesicle coat proteins, is mediated by the ADP-ribosylation factor (Arf) family. Arf proteins have distinct subcellular localizations. ArfGAPs (Arf GTPase-activating proteins) regulate Arf GTPase activity. Thus, each ArfGAP is distinctly localized to allow it to maintain a specific interaction with its target Arf(s). However, the domains that regulate the subcellular localization of ArfGAPs and the way in which these subcellular localizations affect the target specificities of ArfGAPs remain unclear. Recently, we identified two novel ArfGAPs, SMAP1 (Small ArfGAP protein 1) and SMAP2. In the current study, we identified sequences in the carboxy-terminal region of SMAP2 that are critical for its specific subcellular localization and its specificity for Arf proteins.     

A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay

A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O₂ consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.     

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O₂ uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.     

Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential

An efficient system for the import of newly synthesized proteins into highly purified rat liver peroxisomes was reconstituted in vitro. 35S- Labeled acyl-CoA oxidase (AOx) was incorporated into peroxisomes in a proteinase K-resistant fashion. This import was specific (did not occur with mitochondria) and was dependent on temperature, time, and peroxisome concentration. Under optimal conditions approximately 30% of [35S]AOx became proteinase resistant. The import of AOx into peroxisomes could be dissociated into two steps: (a) binding occurred at 0 degrees C in the absence of ATP; (b) translocation occurred only at 26 degrees C and required the hydrolysis of ATP. GTP would not substitute for ATP and translocation was not inhibited by carbonylcyanide-m-chlorophenylhydrazone, valinomycin, or other ionophores.     

Activation of tobacco leaf polyphenol oxidase by sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10 degrees C, suggesting that stabilization of the structure had been improved by the formation of complex.