Mitochondrial and peroxisomal ascorbate peroxidase of pea leaves

The isoenzyme pattern and the substrate specificity of the membrane-bound mitochondrial and peroxisomal ascorbate peroxidases (APX; EC 1.11.1.11) from pea leaves are studied. The substrate specificity of both APXs was assayed using the electron donors ascorbate and pyrogallol, whereas o-dianisidine, hydroquinone, tetramethylbenzidine and 4-methoxy-α-naphthol were also assayed with mitochondrial APX (mitAPX). In leaf mitochondria, the specific activity of APX was similar with pyrogallol and ascorbate, the activity being inhibited by p-CMS. mitAPX showed low activity with the guaiacol peroxidase (GPX)-type substrates, tetramethylbenzidine and 4-methoxy-α-naphthol. Activity of mitAPX with hydroquinone suggest a potential role of mitAPX in the drainage of electrons from the mitochondrial electron chain at the level of ubiquinone. In peroxisomes, the APX (perAPX) specific activity was much higher with pyrogallol than with ascorbate. This perAPX was more sensitive to incubation with Triton X-100 than the mitAPX. By native PAGE the mitAPX was resolved in 6 isoenzyme bands, and the activity of the 3 main bands (mitAPX III, III′ and IV) was inhibited by p-CMS. These 3 major isozymes were also present in mitochondrial membrane fractions. Staining for GPX activity with 4-methoxy-α-naphthol revealed that the APX detected in mitochondria did not have the capacity to oxidize 4-MN, and therefore cannot be considered as true GPX. When intact peroxisomes and peroxisomal membranes were subjected to native PAGE, no APX activity could be detected and this was probably due to the inactivation of perAPX. Results obtained suggest that pea mitochondrial APX (mitAPX) represent a distinct and novel isozyme different from those APXs of chloroplast and cytosolic origin previously reported. The peroxisomal APX (perAPX), however, appears to ressemble the chloroplast APXs as regards its sensitivity to Triton X-100.     

Slipped-strand mispairing in a plastid gene: rpoC2 in grasses (Poaceae)

An exception to the generally conservative nature of plastid gene evolution is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous work by others has shown that maize and rice have an insertion in the coding region of rpoC2, relative to spinach and tobacco. To assess the distribution of this extra coding sequence, we surveyed a broad phylogenetic sample comprising 55 species from 17 angiosperm families by using Southern hybridization. The extra coding sequence is restricted to the grasses (Poaceae). DNA sequence analysis of 11 species from all five subfamilies within the grass family demonstrates that the extra sequence in the coding region of rpoC2 is a repetitive array that exhibits more than a twofold increase in nucleotide substitution, as well as a large number of insertion/deletion events, relative to the adjacent flanking sequences. The structure of the array suggests that slipped-strand mispairing causes the repeated motifs and adds to the mechanisms through which the coding sequence of plastid genes are known to evolve. Phylogenetic analyses based on the sequence data from grass species support several relationships previously suggested by morphological work, but they are ambiguous about broad relationships within the family.      

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism.     

Plant peroxisomes,reactive oxygen metabolism and nitric oxide

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family

Human histatins are a family of low-M(r), neutral to very basic, histidine-rich salivary polypeptides. They probably function as part of the nonimmune host defense system in the oral cavity. A 39-kb region of DNA containing the HIS1 and HIS2 genes was isolated from two human genomic phage libraries as a series of overlapping clones. The nucleotide sequences of the HIS1 gene and part of the HIS2(1) gene were determined. The transcribed region of HIS1 spans 8.5 kb and contains six exons and five introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity, with exon sequences exhibiting 95% identity. The two loci probably arose by a gene duplication event approximately 15-30 Mya. The HIS1 sequence data were also compared with that of STATH. Human statherin is a low-M(r) acidic phosphoprotein that acts as an inhibitor of precipitation of calcium phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81% sequence identity in intron DNA and 80%-88% sequence identity in noncoding exons but only 38%-43% sequence identity in the protein-coding regions of exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong to a single gene family exhibiting accelerated evolution between the HIS and STATH coding sequences.