Takahiro Ishikawa

cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3′-coding regions

Two cDNA clones encoding stromal (SAP28) and thylakoid‐bound (SAP22) ascorbate peroxidase were isolated from a spinach cDNA library constructed by greening cotyledons. The SAP22 and SAP28 contained an open reading frame encoding mature protein of 295 and 345 amino acids with calculated molecular mass of 32 239 Da and 37 710 Da, respectively, preceded by the common transit peptides of 70 amino acid residues. Interestingly, the N‐terminal 364 amino acids of SAP22 were 100% identical with SAP28 except for one C‐terminal amino acid residue (Asp‐365), and the C‐terminal of SAP22, which is the putative transmembrane segment, was 50 amino acids longer than that of SAP28.

Cloning and expression of cDNA encoding a new type of ascorbate peroxidase from spinach

A cDNA clone (SAP1) encoding a peroxidase was isolated from a spinach cDNA library using monoclonal antibodies raised against Euglena ascorbate peroxidase. The deduced amino acid sequence of SAP1 had higher homology with the cytosolic ascorbate peroxidases from plant sources than with bacterial peroxidases and classical plant peroxidases. The peroxidase activity of recombinant SAP1 protein expressed in E. coli was 1.6‐fold higher with ascorbate than with guaiacol, which was similar to those of endogenous cytosolic ascorbate peroxidases. Here we conclude that SAP1 belongs to new type of ascorbate peroxidase from spinach.

Hydrogen peroxide generation in organelles of Euglena gracilis

Abstract Hydrogen peroxide (H 2 O 2 ) generation in intact mitochondria and chloroplasts was investigated in Euglena gracilis . In mitochondria, the addition of respiratory substrate caused the formation of H 2 O 2 . Lactate, the most effective substrate, yielded H 2 O 2 at the rate of 9.5 nmol min −1 mg protein −1 . Chloroplasts showed a rate of H 2 O 2 generation of 5 μmol mg chlorophyll −1 hr −1 under illumination. The results reported here demonstrate that H 2 O 2 is generated in mitochondria and chloroplasts and immediately diffuses from each organelle into the cytosol.

Molecular characterization of Euglena ascorbate peroxidase using monoclonal antibody.

Ascorbate peroxidase (EC 1.11.1.11) has been purified to electrophoretic homogeneity from Euglena gracilis Z. The enzyme showed a molecular mass of 58 kDa on SDS-PAGE and gel filtration, indicating that Euglena ascorbate peroxidase exists as a monomeric form. The substrate specificity for an electron donor and the stability of the purified enzyme were similar to those of cytosolic isozymes from higher plants. One of the characteristic properties was that Euglena ascorbate peroxidase reduces organic hydroperoxides as well as hydrogen peroxide. The N-terminal amino-acid sequence showed no significant similarity to any other ascorbate peroxidase from higher plants. However, the sequence of the peptides from the purified enzyme exhibited a high degree of homology to sequences of cytosolic and chloroplastic ascorbate peroxidases. Monoclonal antibodies against the purified Euglena ascorbate peroxidase were prepared. Two monoclonal antibodies (EAP1 and EAP2) showed high homology to cytosolic ascorbate peroxidases of higher plants, as judged by Western blot analysis. The EAP1 was also specific for chloroplastic ascorbate peroxidase from spinach. These findings indicate that Euglena ascorbate peroxidase exists in highly homologous regions with the ascorbate peroxidases of higher plants.

Purification and characterization of cytosolic ascorbate peroxidase from komatsuna (Brassica rapa)

Abstract In komatsuna ( Brassica rapa ) leaves, the cytosolic ascorbate peroxidase was the major enzyme, but the chloroplastic stromal ascorbate peroxidase accounted for approximately 20% of the total activity, judging from the separate assay of ascorbate peroxidase isozymes reported by Amako et al. [11]. The cytosolic ascorbate peroxidase was purified to electrophoretic homogeneity from komatsuna leaves. The purified enzyme was a monomer with a molecular mass of 28 kDa using gel filtration and SDS-PAGE. The enzyme activity was maximal at pH 6.5 and 38°C, and stable between pH 6.5 and pH 7.5 to 35°C. The inhibition of the enzyme by cyanide and azide showed that it is a hemoprotein. This enzyme was not a glycoprotein. When the enzyme was diluted with the ascorbate-depleted medium, the half inactivation time was approximately 80 min. The komatsuna cytosolic ascorbate peroxidase cross-reacted by western blotting with the monoclonal antibody raised against Euglena cytosolic ascorbate peroxidase.

Requirement for iron and its effect on ascorbate peroxidase in Euglena gracilis

Abstract Euglena gracilis shows an absolute requirement for iron for growth. Iron depletion causes Euglena cells to arrest cell division and the provision of iron allows the iron-deficient cells to return to the normal state. Iron-deficient cells exhaustively take up iron within 1 h of the addition of iron. Incorporated iron exists in a bound form, but not in a free form. The ascorbate peroxidase activity is not found in iron-deficient cells. Lipid peroxides (thiobarbituric acid-reactive substances, TBARS) in iron-deficient cells is much higher than those in iron-sufficient cells. The experimental results reported here suggest that iron is involved in the expression of ascorbate peroxidase activity and Euglena ascorbate peroxidase may possess a second important function in the form of a lipid peroxide-scavenging system, in addition to its action of destroying hydrogen peroxide.

Traffic Sign Recognition Utilizing an Eigen Space Method Based on the KL Transform

This study explains that a method utilizing the eigen spaces obtained by the KL transform for automatic recognition by camera of the speed on a speed limit sign has the following advantages: it is robust in response to changes in intensity patterns caused by the direction the sign is facing and by the amount of light striking the sign, and it is able to reduce the recognition processing time by reducing the number of feature vector dimensions during analysis. The method for recognition of traffic signs previously proposed by the authors of this study was a method for recognition based on extracting geometric shapes from the sign and recognizing them based on their aspect ratios. As such, this method was not able to identify the numbers on a speed limit sign, all of which have identical aspect ratios. It will be shown that the method in this study is able to recognize nearly all speed limits indicated on traffic signs within several 100s of ms after image acquisition. This method was applied to still images and its effectiveness was verified from the perspective of the following requirements for providing accurate information concerning the vehicle surroundings to the driver: high processing speed, high recognition accuracy, detection of all detectable objects without omission, and robustness in response to changes in the surrounding environment.

Method for recognition of numbers on speed limit signs utilizing an eigen space method based on the KL transform

This study explains that a method utilizing the eigen spaces obtained by the KL transform for automatic recognition by camera of the speed on a speed limit sign has the following advantages: it is robust in response to changes in intensity patterns caused by the direction the sign is facing and by the amount of light striking the sign, and it is able to reduce the recognition processing time by reducing the number of feature vector dimensions during analysis. The method for recognition of traffic signs previously proposed by the authors of this study was a method for recognition based on extracting geometric shapes from the sign and recognizing them based on their aspect ratios. As such, this method was not able to identify the numbers on a speed limit sign, all of which have identical aspect ratios. It will be shown that the method in this study is able to recognize nearly all speed limits indicated on traffic signs within several 100s of ms after image acquisition. This method was applied to still images and its effectiveness was verified from the perspective of the following requirements for providing accurate information concerning the vehicle surroundings to the driver: high processing speed, high recognition accuracy, detection of all detectable objects without omission, and robustness in response to changes in the surrounding environment and to geometric changes in the sign image as the vehicle approaches it.