Studies on Iviechanisivis of Cellular Immunity. Part 3
Substrate concentration.-The activity of all enzymes increased as substrate concentrations were raised from 2 to 102 JLM per ml. No substrate inhibition occurred within the limits measured.
Cofactor studies.-The presence of various substances such as magnesium ions, citrate, hyaluronic acid and bovine serum albumin had some effect on the various enzyme systems. Magnesium ions were stimulatory for both phosphatases with maximum stimulation occurring at 3 X 10-5 M:. Beta glucuronidase activity was stimulated by hyaluronic acid (0.008 to 0.04 mg per ml), sodium chloride (0.2 mg per ml) and bovine serum albumin (0.005 to 0.05 mg per ml). Heparin (0.06 to 50 mg per ml) had no effect, while citrate (0.05 to 1.0 mg per ml) was slightly inhibitory.
Experiments with piromen. t- The results in table 3 indicate that both acid phosphatase and beta glucuronidase were inhibited by trace amounts of piromen. Beta glucuronidase activity was depressed 58% by 8.2 JLg piromen per ml and acid phosphatase was depressed 35% by 7.5 JLg. To a slight degree alkaline phosphatase was both stimulated and inhibited.
Experiments with lipopolysaccharides. -The results in tables 4 and 5 indicate that the lipopolysaccharides of several different organisms exerted no appreciable effect on the 3 hydrolases, even though quantities up to 50 JLg per ml were used. Some depression of acid phosphatase activity occurred with highly purified material, but otherwise, when slight stimulation or inhibition was noted, repetitive experiments did not confirm the results.
Experiments with bacterial particles. A particulate fraction prepared from disrupted P. aeruginosa was found to be inhibitory to each enzyme . Alkaline phosphatase and beta glucuronidase were depressed 30% or more by 38 JLg N per ml, while acid phosphatase was depressed 15% by 17.5 JLg N per ml. In vitro experiments with P. aeruginosa.
Acid phosphatase was depressed, alkaline phosphatase was stimulated, and beta glucuronidase was unaffected. Since P. aeruginosa contains neither alkaline phosphatase nor beta glucuronidase, it was possible to carry out similar experiments with live organisms. Alkaline phosphatase was also stimulated when monocytes were incubated with live pseudomonads. The stimulatory response was partially reversed when the concentration of bacteria was increased. Beta glucuronidase was inhibited only by living P. aeruginosa.
In vivo experiments with P. aeruginosa.- When mouse monocytes were allowed to ingest dead P. aeruginosa in vivo, the results obtained were nearly identical to those of previous experiments where no phagocytosis occurred. The results in table 9 indicate that beta glucuronidase activity was depressed. The results of experiments in which monocytes were infected in vivo with live organisms can be found in table 10. The pattern with acid and alkaline phosphatase remained the same as in other experiments. However, the response of beta glucuronidase in infected monocytes was variable.