Molecular characterization of two lipoxygenases from barley.

Two full-length lipoxygenase cDNA sequences (LoxB and LoxC) from barley (Hordeum distichum cv. L. Triumph) are described. The cDNAs share high homology with the barley LoxA cDNA. Southern blotting experiments indicate single copy numbers of the three lipoxygenase genes. RFLP mapping revealed the presence of single lipoxygenase loci. LoxA and LoxB map on chromosome 4 and LoxC on chromosome 7. Two isoenzymes, LOX1 and LOX2, have been purified previously from germinating barley and characterized. LOX1 is encoded by LoxA, while LOX2 is encoded by LoxC. The product related to the third cDNA (loxB) has not been identified so far, suggesting a low protein abundance for the corresponding isoform in barley. Transcripts corresponding with these LOX genes are predominantly observed in grain and in seedling, whereas transcripts corresponding to LoxB and LoxC are also observed in mature vegetative tissue. No lipoxygenase mRNA could be detected in aleurone layer of germinating grain. No significant differences in lipoxygenase mRNA levels were observed in developing grains grown under dormant or non-dormant conditions, suggesting that LOX is not directly involved in induction of grain dormancy.

Differential Expression of Lipoxygenase Isoenzymes in Embryos of Germinating Barley.

Expression of lipoxygenase was studied in barley (Hordeum distichum L.) embryos during germination. Total lipoxygenase activity was high in quiescent grains, dropped during the 1st d of germination, and subsequently increased to a level similar to that in quiescent grains. The contribution of two isoenzymes, lipoxygenases 1 (LOX-1) and 2 (LOX-2), was studied at the activity, protein, and mRNA levels. Activity ratios of the two isoforms were determined via the ratio of 9- and 13-hydroperoxides, which are formed from linoleic acid. Isoenzyme protein levels were determined using specific monoclonal antibodies. mRNA levels were studied using the specific cDNA probes LoxA and LoxC, which correspond to LOX-1 and LOX-2, respectively. The major difference in temporal expression of LOX-1 and LOX-2 was observed in quiescent grains. At this stage, LOX-1 contributed almost exclusively to total lipoxygenase activity. LOX-2 activity rapidly increased until d 2 of germination. From this time point onward, LOX-1 and LOX-2 showed similar patterns at both activity and protein levels. The tissue distribution of the two isoenzymes in the germinating embryo was closely similar, with the highest expression levels in leaves and roots. The levels of LOX-1 and LOX-2 may be regulated mainly pretranslationally, as suggested by the similarity of the protein and mRNA patterns corresponding to the two isoforms.

Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence.

A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded polypeptide was calculated to be 96.392. Its amino acid sequence shows a high homology with that of other plant lipoxygenases identified to date.

Purification and characterization of two lipoxygenase isoenzymes from germinating barley.

Two lipoxygenase isoenzymes (linoleate: oxygen oxidoreductase, EC 1.13.11.12) present in the embryo of germinating barley seed have been purified to homogeneity and characterized. Both isoenzymes are monomeric proteins with a molecular mass of approx. 90 kDa and crossreact on Western blots with antibodies raised against pea lipoxygenase. They have an apparent Km of approx. 16 microM for linoleic acid. The isoenzymes differ in the product formed upon incubation with linoleic acid. One of the isoenzymes (lipoxygenase 1) solely forms the 9-HPOD as a product whereas the 13-HPOD is the major product formed by the other isoenzyme (lipoxygenase 2). Lipoxygenase 1 shows a pH-optimum of 6.5, is active in a broad pH range and has an isoelectric point of 5.2-5.3. Lipoxygenase 2 has the same pH optimum, but is active in a narrow pH range and has a significantly higher pI, namely 6.8-6.9. The occurrence of two isoenzymes was confirmed by peptide analysis of the proteins. Amino acid sequence data obtained from proteolytic fragments of lipoxygenase 1 show up to 50% identity with other plant lipoxygenases.

Permeability properties of peroxisomal membranes from yeasts.

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH1 complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical demonstration of the peroxisomal ATPase of yeasts.

The presence of an ATPase on yeast peroxisomal membranes was studied by immunological methods. Western blot analysis of purified peroxisomal membranes from several yeasts revealed distinct cross-reaction with specific antibodies against the F1-part or the beta-subunit of the mitochondrial ATPase of Saccharomyces cerevisiae. This was not due to mitochondrial contamination as was demonstrated by analytical sucrose gradient centrifugation. Protein A-gold labelling carried out on Lowicryl-embedded methanol-grown Hansenula polymorpha using these antibodies did not result in significant staining. However, when organelles isolated from this yeast were successively incubated with antibodies and protein A-gold prior to embedding, specific labelling was observed on both the peroxisomal membrane and the membrane of damaged mitochondria but not on intact mitochondria. Specific labelling of the peroxisomal membrane was confirmed by freeze-fracture immunocytochemistry. In addition to the peroxisomal membrane, the mitochondrial membrane was also labelled in these experiments. Freeze-fracture immunocytochemistry was also successful for the localization of peroxisomal matrix proteins, e.g. alcohol oxidase and dihydroxyacetone synthase, and of mitochondrial membrane proteins, e.g. cytochrome c oxidase.

A 31P NMR study of the internal pH of yeast peroxisomes.

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by 31P nuclear magnetic resonance (NMR) spectroscopy. 31P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent Pi-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic Pi at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected Pi at a considerably lower pH of approximately 5.8-6.0. Pi at a considerably lower pH of approximately 5.8-6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two Pi-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the Pi-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to Pi in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated Pi was confirmed cytochemically.

A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts.

The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg2+-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent Km for ATP which was 4-6 mM for the peroxisomal ATPase compared to 0.6-0.9 mM for the mitochondrial enzyme. Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl3 and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed in the absence of substrate, in the presence of glycerol 2-phosphate instead of ATP, or in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.