Studies on Iviechanisivis of Cellular Immunity. Part 2
Acid and alkaline phosphatases were assayed by the method of Bessey et al (1946) with pnitrophenyl phosphate as substrate. The resultant p-nitrophenol was quantitatively determined in cell-free supernatants at pH 10 and 420 mu, Alkaline phosphatase was measured in 0.1 M tris buffer, pH 9.1, and acid phosphatase at pH 4.3 and 0.1 M acetate buffer. The level of substrates in reaction mixtures was 5 to 10 ttM per ml. Two ttM of magnesium chloride were added to acid and alkaline phosphatase reaction mixtures whenever in vivo phagocytosis was omitted. Substrates were obtained from the California Corporation for Biochemical Research; p-nitrophenol was obtained from the Nutritional Biochemical Corporation. Nitrogen.-The method of Wong (1923) was used for nitrogen analysis of bacterial particles. Supplements.-Piromen was obtained from Travenol Laboratories, Inc., Morton Grove, Illinois; lipopolysaccharides from Dr. Arthur Johnson, University of Michigan Medical School, and from Difco Laboratories; and polymyxin B from Burroughs Wellcome.
RESULTS
Enzymes.. These studies were extended to determine the site of enzyme activities in sonically disrupted cells. All 3 enzymes were present in the soluble fraction but not in the cellular debris and particulate fractions obtained at 35,000 ref. All enzymes showed greatest activity within the first 12 minutes after addition of substrate. As the time of incubation increased, the rate of hydrolysis decreased.
Effect of pH.–Beta glucuronidase and acid phosphatase activities were noted from pH 3.5 to 5.2. Alkaline phosphatase was active within a narrower range. The optimal pH for beta glucuronidase was 3.9 and for acid and alkaline phosphatases 4.3 and 9.1, respectively. Temperature studies.-Beta glucuronidase activity was obtained at all temperatures between 4 and 56 C with maximum activity at 37 C. Activity was slight at 4 C, while temperatures above 37 C proved suboptimal. The results differed with the phosphatases in that they were inactive at 4 C and temperatures above 37 C did not affect them. Acid phosphatase values at 45 and 56 C were slightly higher than values obtained at 37 C, while alkaline phosphatase activity was approximately the same at all 3 temperatures.
Cell concentration.- beta glucuronidase activity can be detected in preparations containing as few as 5 X 104 cells per ml. As the cell concentration was increased enzymatic activity also increased. Similar studies with the phosphatases indicated that activity was not detectable unless 5 X 106 cells per ml were used. In every case, acid phosphatase was more active than alkaline phosphatase.