RESUMO
Eukaryotic elongation factor 2 (eEF-2) can undergo ADP-ribosylation in the absence of diphtheria toxin. The binding of free ADP-ribose and endogenous transferase-dependent ADP-ribosylation were distinct reactions for eEF-2, as indicated by different findings. Incubation of eEF-2 tryptic fragment 32/33 kDa (32F) with NAD was ADP-ribosylated and gave rise to the covalent binding of ADP-ribose to eEF-2. 32F was revealed to be at the C-terminal by Edman degradation sequence analysis. In our study, the elution of 32F from SDS-PAGE was ADP-ribosylated both in the presence and absence of diphtheria toxin. These results suggest that endogenous ADP-ribosylation of 32F might be related to protein synthesis. This modification appears to be important for the cell function.
Assuntos
Animais , Ratos , ADP Ribose Transferases , Adenosina Difosfato Ribose/metabolismo , Glicosilação , Toxinas Bacterianas/metabolismo , Fragmentos de Peptídeos/metabolismoRESUMO
Poly-ADP-ribosylation of cellular proteins is involved with radiation induced damage and its repair. It has been observed that suspension of human kidney T1-cells in vitro attained elevated levels of poly-ADP-ribosylation due to experimental manipulations necessary for preparation of single cell suspension from monolayer cell cultures. These cells in suspension were exposed to various doses of gamma-rays with or without subsequent repair incubation. The PADPR of histones H3, H1 and H2B increased with increasing dose of radiation and decreased after 90 min or repair incubation. Concomitant with these changes, the affinity of histones to DNA in chromatin reduced immediately after irradiation. Normal affinity was reestablished after post-irradiation repair incubation. The results indicate that induction of poly-ADP-ribosylation of histone proteins by radiation and by manipulations to prepare single cell suspension involved different cellular components.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Linhagem Celular , Radioisótopos de Césio , Cromatina/isolamento & purificação , Raios gama , Histonas/metabolismo , Humanos , Rim , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
ADP-ribosylation (ADPR) of proteins has been shown to be involved with a variety of cellular responses in which chromatin organization and functions are affected. In order to look into this response, human kidney T1-cells were exposed in vitro to various doses up to 3 Gy of 6 MeV neutrons and compared with the effect caused by gamma photons. Whereas in case of neutrons the maximal inhibition of ADPR was reversed at 0.37 Gy, that in case of gamma-rays occurred at 1.5 Gy. For the reversal of inhibition of ADPR of proteins in T1-cells, neutrons were about 4-fold more efficient as compared to gamma rays.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Linhagem Celular , Nêutrons Rápidos , Raios gama , Humanos , Rim/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas/metabolismoRESUMO
Human kidney T1-cells in culture respond to various externally applied stresses, such as mechanical manipulations, trypsinization combined with agitation and centrifugation, Gamma-irradiation, and hyperthermia, by increasing the ADP-ribosylation (ADPR) of proteins in cytoplasm and nuclei. On the other hand, a strong static magnetic field or gravitational force reduces ADPR of proteins. These responses are easily detected in cells in monolayer, yet are masked when the conventional assay of ADPR is used on cells in suspension. The multitude of stimuli for either increasing or depressing ADPR of proteins including histones suggests a common pathway of triggering ADPR in which cellular membranes may be involved.