Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Tenberge, Klaus B.; Ruholl, C.; Heinze, Michael; Eising, Rainer

doi:10.1007/bf01279563

Cited by 14 publications

(12 citation statements)

References 42 publications

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“…In the SDS–PAGE system applied, the apparent molecular mass of the recombinant HNNCATA3 produced in insect cells was approximately 55.0 kDa. We did not observe the higher apparent molecular mass of 59 kDa that was reported previously(Tenberge et al . 1997; Heinze & Gerhardt 2002).…”

Section: Resultscontrasting

confidence: 91%

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Molecular identification, heterologous expression and properties of light‐insensitive plant catalases

Engel

Schmidt

Feierabend

2005

Plant Cell & Environment

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Most catalases are inactivated by light in a heme-sensitized and O 2 -dependent reaction. In leaves of the alpine plant Homogyne alpina and in the peroxisomal cores of Helianthus annuus , light-insensitive catalases were observed. For the catalases Hacat1 of H. alpina and HnncatA3 of H. annuus , cDNA clones were obtained. Expression of recombinant active enzymes in insect cells confirmed that they coded for light-insensitive catalases. Kinetic and catalytic properties of light-sensitive or light-insensitive catalases did not differ substantially. However, the specific activity of the latter was markedly lower. The light-insensitive catalase HaCAT-1 was not resistant against inactivation by superoxide. Amino acid sequences of the light-insensitive catalases HaCAT-1 and HNNCATA3 were highly identical. They showed only a few exceptional amino acid substitutions at positions that are highly conserved in other catalases. These appeared to be localized mainly in a surface cavity at the entrance of a minor channel leading to the central heme, suggesting that this region played some, though yet undefined, role for light sensitivity. While the replacement of a highly conserved His by Thr225 was the most unique substitution, a single exchange of His225 by Thr in the light-sensitive catalase SaCAT-1 by mutagenesis was not sufficient to reduce its sensitivity to photoinactivation.

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Section: Resultscontrasting

confidence: 91%

“…(1992). A peroxisomal core fraction was prepared from sunflower cotyledons according to Tenberge et al . (1997).…”

Section: Methodsmentioning

confidence: 99%

Molecular identification, heterologous expression and properties of light‐insensitive plant catalases

Engel

Schmidt

Feierabend

2005

Plant Cell & Environment

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“…9). We have observed smaller crystal unit cells in CAT crystalls in transgenic tobacco than, e.g., Tenberge et al (1997) in sunflower cotyledons or Sato et al (1993) in beef liver. However, formation of different types of cores suggests that differences in the molecular structure of CAT might be also responsible for the capability of one CAT type to build different crystals (Harris and Holzenburg 1995).…”

Section: Discussionmentioning

confidence: 86%

“…However, formation of different types of cores suggests that differences in the molecular structure of CAT might be also responsible for the capability of one CAT type to build different crystals (Harris and Holzenburg 1995). Tenberge et al (1997) proved that PC have CAT activity and that the enzymatic activity of CAT was barely affected by its integration into the cores. Kleff et al (1997) proved that 59 and 55 kD CAT isozymes in peroxisomes of sunflower cotyledons were rather specifically divided into the core and matrix, respectively.…”

Section: Discussionmentioning

confidence: 97%

“…Purification of peroxisomal cores: Peroxisomal cores (PC) were purified by Percoll density gradient centrifugation of crude particle fractions from tobacco leaves according to the method of Tenberge et al (1997). To prepare a crude particle fraction, 200 g of tobacco leaves were homogenized in the extraction medium (0.3 M mannitol, 20 mM pyrophosphate buffer (pH 7.5), 1 mM EDTA, 0.1 % BSA), and after filtration centrifuged at 1 000 g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

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Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

Synková¹,

Semorádová²,

Schnablová³

et al. 2006

Biol. plant.

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Cytokinin (CK) content and activities of several antioxidant enzymes were examined during plant ontogeny with the aim to elucidate their role in delayed senescence of transgenic Pssu-ipt tobacco. Control Nicotiana tabacum L. (cv. Petit Havana SR1) and transgenic tobacco with the ipt gene under the control of the promoter of small subunit of Rubisco (Pssu-ipt) were both grown either as grafts on control rootstocks or as rooted plants. Both control plant types showed a decline in total content of CKs with proceeding plant senescence. Contrary to this both transgenic plant types exhibited at least ten times higher content of CKs than controls and a significant increase of CK contents throughout the ontogeny with maximal values in the later stages of plant development. Significantly higher portion of O-glucosides was found in both transgenic plant types compared to control ones. In transgenic plants, zeatin and zeatin riboside were predominant type of CKs. Generally, Pssu-ipt tobacco exhibited elevated activities of antioxidant enzymes compared to control tobacco particularly in the later stages of plant development. While in control tobacco activity of glutathione reductase (GR) and superoxide dismutase (SOD) showed increasing activity up to the onset of flowering and then gradually decreased, in both transgenic types GR increased and SOD activity showed only small change throughout the plant ontogeny. Ascorbate peroxidase (APOD) was stimulated in both transgenic types. The manifold enhancement of syringaldazine and guaiacol peroxidase activities was observed in transgenic grafts throughout plant ontogeny in contrast to control and transgenic rooted plants, where the increase was found only in the late stages. Electron microscopic examination showed higher number of crystallic cores in peroxisomes and abnormal interactions among organelles in transgenic tobacco in comparison with control plant. The overproduction of cytokinins resulted in the stimulation of activities of AOE throughout the plant ontogeny of transgenic Pssu-ipt tobacco.Additional key words: ascorbate peroxidase, catalase, glutathione reductase, guaiacol peroxidases, superoxide dismutase, syringaldazine peroxidase.⎯⎯⎯⎯ sequence of the gene coding for small subunit of Rubisco; SPOD -syringaldazine peroxidase; SOD -superoxide dismutase; T -transgenic rooted plants; T/C -transgenic grafts; TEM -transmission electron microscopy.

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High Resolution Scanning Electron Microscopy of Protein Inclusions (Cores) purified from Peroxisomes of Sunflower (Helianthus annuus L.) Cotyledons

Heinze

Reichelt

Kleff

et al. 2000

Cryst. Res. Technol.

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This paper describes the first characterization of protein inclusions (so‐called cores) built in higher plant peroxisomes by high resolution scanning electron microscopy (HRSEM). Purification of peroxisomal inclusions from sunflower (Helianthus annuus L.) cotyledons led to highly enriched preparations suitable for HRSEM. Isolated peroxisomal cores occurred mostly as quadrangular blocks, but sometimes also cube‐like particles were found. In all cases, two edges of the blocks had similar lengths resulting in two square sides. The third edge was shorter than the two edges of the square sides. Edge lengths of the square side were typically in the range from 200 to 600 nm, however, also values of up to 1 μm were observed. Measurements of the three edge lengths of individual cores allowed for the first time to determine the volume of peroxisomal cores from plants. Core volumes ranged from about 0.01 μm3 to almost 0.1 μm3 indicating that cores represent a significant part of the total peroxisomal volume. As revealed by HRSEM, the surface layers of cores were built of regularly arranged, repeating square units with an edge length of about 20 nm. Between these units, interstices of about 4 nm appeared. Light optical diffraction analysis of HRSEM micrographs of cores revealed diffraction patterns up to the second order indicating that peroxisomal cores grown in vivo have a high degree of regularity. HRSEM on immunogold‐labelled samples verified that isolated cores contained the peroxisomal enzyme catalase. In summary, the results suggest cores to be formed in sunflower peroxisomes by crystallization of a specific molecular form of catalase, which is the predominant protein component of the particles. Another form of catalase, differing in amino acid sequence from core catalase, obviously does not participate in the crystallization process, although it is present in the peroxisomal matrix during the formation of cores in vivo.

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Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Cited by 14 publications

References 42 publications

Molecular identification, heterologous expression and properties of light‐insensitive plant catalases

Molecular identification, heterologous expression and properties of light‐insensitive plant catalases

Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny

High Resolution Scanning Electron Microscopy of Protein Inclusions (Cores) purified from Peroxisomes of Sunflower (Helianthus annuus L.) Cotyledons

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