Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperatures near the main bilayer phase transition and saturated long-chain diacylglycerol in the bilayer modulate the effectiveness of the reaction products. The purpose of this study was to identify possible mechanisms for these effects of temperature and diacylglycerol. Various fluorescent probes were used to asses changes in the ability of the reaction products to perturb the bilayer and promote enzyme binding to he membrane surface. Temperature appeared to cause three effects. First, the degree of binding of enzyme at the end of the latency period was greatest near the phase transition temperature where the latency was shortest. Second, the bilayer was more sensitive to perturbation by reaction products near the transition. Third, the disturbance provoked by the products was confined to the membrane surface below the transition but affected deeper regions at higher temperature where the latency period was greater. The latter two effects of temperature required the presence of calcium. Diacylglycerol promoted lateral segregation of reaction products in the bilayer. This effect corresponded with the tendency of diacylglycerol to reduce the length of the latency period at temperature below the phase transition. Therefore, it appeared that temperature affects the latency period by alternating the binding of the enzyme and the depth and magnitude of the bilayer perturbation caused by reaction products. Alternatively, diacylglycerol may enhance the effectiveness of reaction products by inducing them to segregate in the bilayer and thus create local regions of increased impact on the bilayer surface.

The effect of temperature and various glycerides to modulate the ability of lysolecithin and fatty acid to promote high phospholipase A2 activity was studied using dipalmitoylphosphatidylcholine large unilamellar vesicles as substrate. The length of the lag phase prior to the accumulation of sufficient hydrolysis products (lysolecithin and fatty acid) to support high phospholipase activity was shortest at temperatures near the thermotropic phase transition of the phospholipid substrate. A reduction in the lag phase correlated with a reduction in the requirement for hydrolysis products at the phase transition temperature, where the bilayer exists in a state of fluctuating domains of gel and liquid crystal. Dipalmitoylglycerol and tripalmitoylglycerol also reduced the length of the lag phase. This reduction was both concentration-dependent and temperature-dependent relative to the phase transition in the presence of the glycerides. As with the effect of temperature, the ability of di- and triglycerides to decrease the lag time correlated with a decrease in the amount of reaction products necessary to promote high phospholipase activity. This effect coincided with the tendency of the glycerides to form domains in the bilayer. Glycerides that did not form domains either had no effect (monopalmitoylglycerol) or increased the length of the lag phase (dicaprylglycerol). These data suggest that the effect of the reaction products to increase phospholipase A2 activity is aided by the presence of fluctuations in lipid domains within the bilayer.

The activity of phospholipase A2 from snake venom to hydrolyze bilayers of phosphatidylcholines is greatly enhanced by the presence of the hydrolysis products, lysolecithin and fatty acid, in the bilayer. The fluorescence of several probes of membrane structure was used to monitor changes in bilayer physical properties during vesicle hydrolysis. These changes were compared to emission spectra and fluorescence polarization results occurring upon direct addition of lysolecithin and/or fatty acid to the bilayer. The excimer to monomer ratio of 1,3-bis(1-pyrene)propane was insensitive to vesicle hydrolysis, suggesting that changes in the order of the phospholipid chains were not relevant to the effect of the hydrolysis products on phospholipase activity. The fluorescence of 6-propionyl-2-(dimethylamino)-naphthalene (Prodan) suggested that the polarity of the bilayer in the region of the phospholipid head groups increases as the hydrolysis products accumulate in the bilayer. The fluorescence of 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) confirmed that such effects were restricted to the bilayer surface. Furthermore, the lysolecithin appeared to be the product most responsible for these changes. These results suggested that lysolecithin increases the activity of phospholipase A2 during vesicle hydrolysis by disrupting the bilayer surface, making the phospholipid molecules more accessible to the enzyme active site.

An enzyme immunoassay was set up with the aim of determining the serum levels of Müllerian inhibiting substance (MIS) during childhood. A monoclonal antibody against purified bovine MIS was combined with a polyclonal antibody against recombinant human MIS to make a sandwich assay. This assay detected MIS in human serum within the following criteria. Ninety-eight boys, aged between birth and 18 yr, who had been admitted to the Royal Children's Hospital, were included. MIS levels were measured in samples taken for biochemical screening of unrelated disorders. MIS was detected in the serum up to 16 yr of age, but was low beyond 12 yr and undetectable at 18 yr. High MIS levels were found at 4-12 months, consistent with MIS having an important function at this time. Germ cells undergo an important transformation from gonocytes to spermatogonia at the same time as the MIS levels peak, suggesting a possible function for MIS.