Phosphatase activity in homogenates and particulates from normal and poliovirus infected tissue cultures
Creators: name: Deig, Elmer Frank
Publisher: name: University of Utah
Year: 2012
Subjects: Analysis
Tissue Culture

description: A study was made on the total ATP-ase, alkaline phosphatase, and glucose-6-phosphatase activity in homogenates prepared from normal and poliovirus infected monkey kidney cells in tissue culture.  A characterization of such activity under standardized assay conditions revealed that in comparable amounts of cell homogenate ATP-ase was 8-10 time as active as alkaline phosphatase and glucose-6-phosphatase.  Substrate hydrolysis was brought about by the activity of each enzyme at a rate which was constant with respect to time.  The amount of substrate hydrolyzed, in each case, was directly proportional to the concentration of homogenate used in the assay system.  Within certain limits this was also true with respect to substrate concentration.  However, with increasing amounts of substrate and a constant amount of cell homogenate a point was reached where the activity of each enzyme became independent of initial substrate concentration. Under one set of assay conditions the total ATP-ase in cell homogenates was shown to be optimally active at approximately pH 7 while under another enzyme showed a nearly directly proportional increase in activity per unit time between pH 4 and pH  10.  Using standardized assay conditions such ATP-ase activity was markedly enhanced over that found in similar systems lacking added calcium and magnesium ion.  No further stimulation of the activity of this enzyme, or of alkaline phosphatase and glucose-6-phosphatase, resulted from their interaction with 2,4-dinitrophenol.  The substrate hydrolysis produced by the activity of each enzyme of cell homogenate preparations resulted from irreversible reactions.  Such homogenates were incapable of carrying out an exchange reaction of radioactive orthophosphate with the phosphorous from any of the substrates used in this study.  Concentrated suspensions of equal numbers of normal and poliovirus infected cells at 37° C were sampled periodically.  Cell homogenates prepared from these samples were analyzed for total nitrogen content and for total ATP-ase, alkaline phosphatase, and glucose-6phosphatase activity.  The homogenates prepared from equal numbers of virus infected cells showed a logarithmic decrease in nitrogen with respect to time over the period from four to twelve hours after infection.  No additional loss in nitrogen occurred during the following twelve hours.  Such nitrogen losses were associated with a decrease in the activity of the three enzymes.  In the case of ATP-ase this decrease between the four and twelve hour period was linear with respect to time.  The activity of this enzyme remained constant during the flowing twelve hours.  The nitrogen content and activity of the three enzymes in homogenates prepared from normal cell suspensions did not increase over the sampling interval.  The large amount of bicarbonate required to maintain the pH of the culture under these conditions produced an unfavorable environment for the cells.  This was confirmed by the finding that normal cells placed initially in tissue culture medium, at standard pH, containing excessive amounts of bicarbonate showed no metabolic activity.  Such cell exhibited a marked reduction in total nitrogen and in the activity of the three enzymes over a sampling interval of twelve hours.  When normal cells were placed in the tissue culture medium, at standard pH, containing such excessive concentrations of bicarbonate, and immediately infected with poliovirus, a delay in the synthesis of, and a marked reduction in final titer of the virus resulted from that found under control conditions.  A crude separation of cell particulates from the homogenate was made by means of ultracentrifugation in a discontinuous sucrose gradient.  The activity of ATP-ase in each fraction was linear with respect to time with maximal activity being found in the mitochondrial fraction.  When such a separation was carried out with homogenates prepared from equal number of normal and poliovirus infected cells following agitation at 37° C for two hours it was found that the agitation procedure brought about significant losses of nitrogen in approximately equal amount from the mitochondrial fraction of both normal and infected cells.  Virus induced losses of cellular nitrogen occurred primarily in the nuclear and microsomal fractions.  Both types of nitrogen loss were correlated with decrease ATP-ase activity in the respective particulate fractions.