Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes

The presence of endogenous phospholipase A (PL-A) activity of U. urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas. Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated. The pH optimum was about 8.5-9 for phospholipase A(1) (PL-A(1)) in the three serovars. A more acidic pH optimum of 6 was observed for phospholipase A(2) (PL-A(2)) enzymes in serovars 3 and 4. However, a very significant stimulation of PL-A(2) activity in serovar 8 occurred around pH 7. The specific activity of PL-A(2) was always 50-100 fold higher than PL-A(1) activity in the pH range studied. Ca2+ ions only slightly stimulated PL-A(1) activity in all 3 serovars. PL-A(2) activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8. Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A(2) activity in serovar 4. EDTA inhibition corresponded to Ca2+ stimulation for PL-A(2) activity for serovars 3 and 8. A general stimulation of PL-A(2) activity by diethyl ether was evident but the degree of stimulation varied with the serovar. Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8. PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme. All 3 serovars were inactivated by heat. A broad pH optimum for PL-C activity was evident around 7-8. Diethyl ether enhanced PL-C activity of serovar 8. Sodium deoxycholate and heat were inhibitory to PL-C activity. The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria. However, the major differences in the specific characteristics of specially PL-A(1) and PL-A(2) suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria. Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival. The ureaplasma PL-As are also markedly different from one serovar to another. This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus. There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta. There are marked differences in calcium concentrations under specific circumstances in various host tissues. Thus the differences in specific activity among the phospholipases of the serovars of U. urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.