Further characterization of a novel triacylglycerol hydrolase activity (pH 6.0 optimum) from microvillous membranes from human term placenta

Abstract

We recently identified the presence of two distinct triacylglycerol hydrolases 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 to lipoprotein lipase, whereas TAG hydrolase with a pH optimum of 6.0 still to be fully characterized. In order to understand the functional and structural relationships between these two TAG hydrolases of MVM we have further investigated their biochemical and molecular properties. The presence of oleic acid inhibited TAG hydrolase activity with a pH optimum of 8.0 by 60 per cent whilst it had very little effect on the pH 6.0 TAG hydrolase activity. 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 were 0.32 and 0.037 nmol oleic acid/min mg/protein. Treatment of MVM with phenylmethylsulphonofluoride or protamine had no effect on TAG hydrolase at pH 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 iodoacetate 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 at 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, whereas membrane associated TAG hydrolase activity with optimum pH 8.0 did not 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. The 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 and had a pH optimum of 6.0. Labelling of samples with [(14)C]tetrahydrolipstatin also confirmed that the TAG hydrolase active protein was a 70 kDa protein. 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 lipoprotein lipase.