Nonetheless, GPD activity was selleck chemical detected almost exclusively in the membrane fraction. Extensive washing of the membrane preparations with increasing concentrations of NaCl (up to 1 M) in 10 mM Tris buffer (pH 7.5) with or without 10 mM EDTA did not affect the levels of GPD activity in the membranes (data not shown), suggesting that the GPD is not a loosely bound membrane protein adsorbed onto the membrane surface. The identification
of the M. hyorhinis GPD was further strengthened by showing its homology to the active GPD of M. pneumoniae and to the GPD of Thermoanaerobacter tengcongensis (GenBank accession no. 2PZ0_A) where strictly conserved residues involved in the activity were identified (Fig. 2, Shi et al., 2008). We suggest that GPD is an essential enzyme in the turnover of glycerophospholipids, the major building blocks of the lipid bilayer of M. hyorhinis membranes. First, the fatty acids are cleaved resulting in the formation of glycerophosphodiesters, which are then further cleaved by GPD to yield glycerol-3-phosphate (Schmidl et al., 2011). Upon incubation
of M. hyorhinis extracts with radiolabeled PG, a decrease in the radioactivity of the PG band with a concomitant increase in the radioactivity of the lysophospholipid and FFA fractions were noticed (data not shown), suggesting a phospholipase activity. The activity was almost exclusively associated with isolated membrane preparations (data not shown). When reaction mixtures containing M. hyorhinis membranes and the fluorescent substrate C12-NBD-PC were incubated for up to 4 h at 37 °C, two fluorescently labeled breakdown products were detected on the TLC plates, the learn more major being C12-NBD-LPC with nonfluorescent fatty acid in position 1 hydrolyzed and the minor C12-NBD-FFA (Fig. 3), suggesting the activity of a PLA in M. hyorhinis membranes. In control experiments, using snake venom PLA2, the breakdown product of C12-NBD-PC was exclusively C12-NBD-FFA. The PLA activity of M. hyorhinis was neither stimulated by Ca2+ (0.1–10 mM) nor inhibited
by EGTA (5 mM) and had a broad pH spectrum (pH 7.0–8.5). Quantitative analysis of the fluorescence Metalloexopeptidase products obtained by the hydrolysis of C12-NBD-PC by M. hyorhinis membranes is shown in Table 1. The ratio of C12-NBD-LPC to C12-NBD-FFA after treatment of C12-NBD-PC with M. hyorhinis membranes was 2.5 after a short incubation period (up to 1 h) and 0.8 after a prolonged incubation period (4 h), suggesting that M. hyorhinis possess a nonspecific PLA activity capable of hydrolyzing both position 1 and position 2 of the C12-NBD-PC, but with a somewhat higher affinity to position 1. The possibility that M. hyorhinis possess a PLA1 (Istivan & Coloe, 2006) or PLA2 (Rigaud & Leblanc, 1980) as well as a lysophospholipase (Gatt et al., 1982) was excluded as we were unable to demonstrate lysophospholipase activity using C12-NBD-LPC (data not shown). The in silico analysis of M.