Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many ...Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.展开更多
Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterizati...Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.展开更多
文摘Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.
文摘Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.
