Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

The extent of increase in the activity of phenylalanine ammonia-lyase upon illumination for 15 hr of cultured Petroselinum hortense cells was greatly dependent upon the age of the culture. Two distinct peaks in specific activity were observed during the growth cycle, one occurring at the beginning and the other at the end of the period of increase in cell fresh weight. High yields in both cell fresh weight and enzyme activity were obtained with the second peak shortly before the stationary phase of the culture was reached. Cells were harvested at this stage and stored at ?20 °C.The enzyme was purified from the frozen cells to apparent homogeneity by precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose, Sephadex G-200 and hydroxyapatite columns, and preparative polyacrylamide gel electrophoresis. An over-all yield of 16% was achieved with a 440-fold increase in the specific activity by purification of the enzyme through the hydroxyapatite step.Upon analytical polyacrylamide gel electrophoresis either in the presence or in the absence of sodium dodecyl sulfate, the purified protein migrated essentially as a single band. Molecular weights of about 330,000 and 83,000, respectively, were estimated for the enzyme and for its protein subunits. Thus, the enzyme molecule seems to be composed of four probably identical protein subunits. Two Michaelis constants for l-phenylalanine (K_m^L, 3.2 × 10^?5 M, and K_m^H, 2.4 × 10^?4 M) and a Hill coefficient of h = 0.6 were obtained. This suggests that the enzyme is subject to regulation of its catalytic properties by negative cooperativity of the protein subunits.