ABSTRACT
It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.
Subject(s)
Glucosephosphate Dehydrogenase/metabolism , Buffers , Cell Line , Drug Stability , HeLa Cells/enzymology , Humans , Kinetics , NADP , Oxidation-ReductionABSTRACT
A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.
Subject(s)
Glucosephosphate Dehydrogenase/isolation & purification , Cells, Cultured , Glucosephosphate Dehydrogenase/metabolism , Humans , Kinetics , Macromolecular Substances , Molecular WeightABSTRACT
The WI-38 strain of human diploid cells (HDC) and two strains, L-58 and L-63 isolated in Moscow Research Institute of Virus Preparations were examined in the course of sequential passages for the presence of primate oncornaviruses and their antigens. In parallel, the tumorigenic capacity of the cells was tested by inoculating them into the cheek pouch tissue of Syrian hamsters. In all cases the examined HDC cultures contained no oncornavirus particles or their antigens and in no case showed any infectious activity determined in the KC-test. Only on one subculture of the L-63 strain cells at the 44th passage was a positive result obtained in immunoprecipitation test with antiserum to the LPV oncornavirus. Cells of the L-58 strain which had undergone 27 and 43 passages showed tumorigenicity in hamster experiments. In subsequent passages of these HDC strains no such properties were found. The regular control for the absence of oncogenic message is a necessary condition for the use of HDC strains in virological practice.
Subject(s)
Cells, Cultured/microbiology , Diploidy , Retroviridae/isolation & purification , Cell Line , Fluorescent Antibody Technique , Humans , Hybridization, Genetic , Microscopy, Electron , Molecular Biology , RNA-Directed DNA Polymerase/analysis , Retroviridae/enzymology , Retroviridae/pathogenicityABSTRACT
Glucose 6-phosphate dehydrogenase (E. C. 1.1.1.49) was found to be inactivated by 30% for 25 min the incubation at 37 degrees C in extracts of human diploid cells and mouse liver, and the stability of the enzyme was found to reduce in aging of human diploid cells and mouse. The stability of dehydrogenase of mice treated with "geroprotector", 2-ethyl-6-methyl-3-hydroxypyridine, exceeds the control level.
Subject(s)
Glucosephosphate Dehydrogenase/metabolism , Liver/enzymology , Picolines/analogs & derivatives , Aging , Animals , Diploidy , Drug Stability , Humans , Mice , Picolines/pharmacologyABSTRACT
Some properties of 4 diplod cell strains derived from the lung and skin-muscle tissue of human embryos were studied. Only one strain (L-63) was shown to have a stable karyotype throughout its life span and induced no tumor formation in the cheek pouch of hamsters. Species-specific and group antigens were revealed. In the cell strains of the lung and skin-muscle derivation differences in the distribution of lactate dehydrogenase isoenzymes were observed and found characteristic of the original tissue cells.
Subject(s)
Cell Line , Animals , Cricetinae , Diploidy , Epitopes , Humans , Isoenzymes , L-Lactate Dehydrogenase/metabolism , Lung/embryology , Muscles/embryology , Neoplasms, Experimental/etiology , Skin/embryologyABSTRACT
Different strains of diploid cells were studied experimentally in Syrian hamsters by heterotransplantation into cheek pouch tissue for detection of their tumorigenic activity. Cortisone and antilymphocyte serum were used as immunodepressors. The results showed that the use of antilymphocyte serum increased considerably the sensitivity of the method of determination of cell culture tumorigenic activity.
