A simple, rapid and sensitive staining method for detecting the phosphatase in polyacrylamide gels was developed, in which p-nitrophenyl phosphate (pNPP) was used as substrate.This method was based on the activity of ...A simple, rapid and sensitive staining method for detecting the phosphatase in polyacrylamide gels was developed, in which p-nitrophenyl phosphate (pNPP) was used as substrate.This method was based on the activity of p - nitrophenylphosphatase (pNPPase) to release Pi from pNPP within the polyacrylamide gels which then combines with lead ion to form the lead phosphate and precipitates in the gel as white bands. These bands can be changed to dark brown bands on a transparent background due to formation of PbS by treating with (NH4)2S within the gel.The crude extracts of H22a hepatoma ascites cells and normal mouse liver cells were analyzed by this method and found that there are some difference between the two kinds of cells.展开更多
Many reports show that cell carcinogenesis is related to DNA methylation in the eukaryote. DNA of most cancer cells and oncogenes is in undermethylation. DNA methylation level [5mC/(C+5mC)％] is dependent on the DNA m...Many reports show that cell carcinogenesis is related to DNA methylation in the eukaryote. DNA of most cancer cells and oncogenes is in undermethylation. DNA methylation level [5mC/(C+5mC)％] is dependent on the DNA methylase activity that catalyzes the incorporation of methyl group into cytosine of DNA from展开更多
A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful s...A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase are also studied.展开更多
文摘A simple, rapid and sensitive staining method for detecting the phosphatase in polyacrylamide gels was developed, in which p-nitrophenyl phosphate (pNPP) was used as substrate.This method was based on the activity of p - nitrophenylphosphatase (pNPPase) to release Pi from pNPP within the polyacrylamide gels which then combines with lead ion to form the lead phosphate and precipitates in the gel as white bands. These bands can be changed to dark brown bands on a transparent background due to formation of PbS by treating with (NH4)2S within the gel.The crude extracts of H22a hepatoma ascites cells and normal mouse liver cells were analyzed by this method and found that there are some difference between the two kinds of cells.
基金Project supported by the National Natural Science Foundation of China.
文摘Many reports show that cell carcinogenesis is related to DNA methylation in the eukaryote. DNA of most cancer cells and oncogenes is in undermethylation. DNA methylation level [5mC/(C+5mC)％] is dependent on the DNA methylase activity that catalyzes the incorporation of methyl group into cytosine of DNA from
基金Project supported by the National Natural Science Foundation of China.
文摘A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase are also studied.
