Further characterization of a novel triacylglycerol hydrolase activity (pH6.0 optimum) from microvillous membranes from human term placenta

We recently identified the presence of two distinct triacylglycerol hydrola ses with pH optima of 6.0 and 8.0 in human placental microvillous membranes (MVM). The TAG hydrolase with a pH optimum of 8.0 has properties similar t o lipoprotein lipase, whereas TAG hydrolase with a pH optimum of 6.0 still to be fully characterized. In order to understand the functional and struct ural relationships between these two TAG hydrolases of MVM we have further investigated their biochemical and molecular properties. The presence of ol eic acid inhibited TAG hydrolase activity with a pH optimum of 8.0 by 60 pe r cent whilst it had very little effect on the pH 6.0 TAG hydrolase activit y. K-m values for TAG hydrolases at pH 6.0 and pH 8.0 optima were 170.6 and 9.83 nmol triolein, respectively, whereas the corresponding V-max values w ere 0.32 and 0.037 nmol oleic acid/min mg/protein. Treatment of MVM with ph enylmethylsulphonofluoride or protamine had no effect on TAG hydrolase at p H 6.0 whereas both decreased activity at pH 8.0, by 70 per cent and 52 per cent, respectively (P<0.05), compared with control. p-Chloromercuribenzoate inhibited both TAG hydrolase activities by 25-30 per cent whereas iodoacet ate inhibited TAG hydrolase activity with optimum pH 8.0 by 74 per cent and the activity at pH 6.0 by 28 per cent. Unlike the TAG hydrolase activity a t pH 8.0, the activity at pH 6.0 was not affected by heparin. TAG hydrolase activity at pH 6.0 was significantly decreased compared with that of pH 8. 0 optimum TAG hydrolase activity in smokers placenta. A threefold increase in pH 6.0 TAG hydrolase activity was observed following differentiation, wh ereas membrane associated TAG hydrolase activity with optimum pH 8.0 did no t change. The TAG hydrolase with optimum pH 6.0 was subsequently purified from MVM to almost 1000-fold enrichment of the activity over the starting material. Th e final preparation however, still contained three distinct protein bands ( 90, 70 and 45 kDa). When extracted from non-denaturing polyacrylamide gels, the 70 kDa protein was the only protein to have TAG hydrolysing activity a nd had a pH optimum of 6.0. Labelling of samples with [C-14]tetrahydrolipst atin also confirmed that the TAG hydrolase active protein was a 70 kDa prot ein. In conclusion, we report that there is a 70 kDa TAG hydrolase with optimum pH 6.0 in human placental MVM which is quite distinct from placental lipopr otein lipase. (C) 2000 Harcourt Publishers Ltd.