CPP32/Yama/apopain cleaves the catalytic component of DNA-dependent protein kinase in the holoenzyme

AbstractDNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic component (p460) and a DNA-binding component Ku protein. Immunoblot analysis after treatment of Jurkat cells with anti-Fas antibody demonstrated the cleavage of p460 concomitantly with an increase in CPP32/Yama/apopain activity. Recombinant CPP32/Yama/apopain specifically cleaved p460 in the DNA-PK preparation that had been purified from Raji cells into 230- and 160-kDa polypeptides, the latter of which was detected in anti-Fas-treated Jurkat cells. The regulatory component Ku protein was not significantly affected by CPP32/Yama/apopain. DNA-PK activity was decreased with the disappearance of p460 in the incubation of DNA-PK with CPP32/Yama/apopain. These results suggest that the catalytic component of DNA-PK is one of the target proteins for CPP32/Yama/apopain in Fas-mediated apoptosis

Evolutionary Strategies of Highly Functional Catalases for Adaptation to High H₂O₂ Environments

Enzymatic evolutionary strategies for adaptation to a high H2O2 environment have been evaluated using catalases with high catalytic efficiency isolated from two H2O2-tolerant bacteria, Exiguobacterium oxidotolerans and Psychrobacter piscatori. The entrance size of the narrow main channel in catalase has been estimated by determining the formation rate of the intermediate state of peracetic acid (b), which is a larger substrate than H2O2 versus that of catalase activity with H2O2 (a) (calculated as b/a). The ratio of b/a in E. oxidotolerans catalase (EKTA) is much higher than that of P. piscatori catalase (PKTA). To elucidate the structural differences between the catalases, the amino acids present in the main channel have been compared between the two catalases and other catalases in the database. The combination of amino acid residues, which contribute high catalytic efficiency in the narrow main channel of EKTA were different from those in PKTA. In this review, we discuss strategic differences in the elimination of high concentration of H2O2 owing to differences in the phylogenetic positions of catalases. In addition, we describe the relationships between the environmental distributions of genera involved in H2O2-resistant bacteria and their catalase functions based on the main channel structure of catalase

Growth-dependent catalase localization in Exiguobacterium oxidotolerans T-2-2T reflected by catalase activity of cells.

A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2(T), exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state

Inner circumference localization ratio of catalase in strain T-2-2^T cells on the basis of particles in ultrathin sections exhibiting catalase immunolocalization.

<p>Cells from 5 (n = 19), 14 (n = 33) and 24 (n = 47) h cultures are indicated by open, blue filled and red filled circles, respectively. Particles locating within 50 nm from the surface of the cell are considered as surface- localized particles. Black bars indicate the average inner circumference localization ratio in each growth phase.</p

An ultrathin section showing immunolocalization of catalase in <i>E. oxidotolerans</i> T-2-2^T after 5, 14 and 24 h of culture.

<p>The particles show the localization of catalase. Cells from 5, 14 and 24-exponential, early stationary and mid-stationary growth phases, are shown in (a), (b) and (c), respectively. Bar, 1 µm.</p

Effect of pH on activities of extracellular and intracellular catalases of <i>E. oxidotolerans</i> T-2-2^T.

<p>Extracellular and intracellular catalases are indicated by filled and open circles, respectively. The buffers (50 mM) used were as follows: pHs 3.0–6.0, citrate-NaOH; pHs 4.0–5.0, acetate-NaOH; pHs 6.0–8.0, sodium phosphate; pHs 8.0–9.0, Tris-HCl; pHs 9.0–10.0, borate-NaOH. The activities are relative to that at pH 6.5, which is 100%.</p