All assays were done in duplicate. devoid of PLRP2. All of the colipase-dependent activity segregated with the PLRP2-containing fraction, consistent with the conclusion that PLRP2 is the major colipase-dependent lipase in the pancreas of newborns. for 10 min at 4C. The supernatant was harvested for further analysis. Colipase was removed from the extracts of the pancreata of PLRP2-deficient and wild-type mice by dialysis. We demonstrated the efficient removal of colipase by adding an aliquot of the heat-inactivated extract to a Rabbit Polyclonal to ELAV2/4 reaction mixture of pancreatic triglyceride lipase and inhibitory concentrations of taurodeoxycholate as described below. The extract did not restore activity to bile saltCinhibited pancreatic triglyceride lipase, showing that the colipase had been effectively removed. Protein (1 mg) was assayed for lipase activity against trioctanoin emulsified in Pamiparib 4 mmol/L taurodeoxycholate by the pH-stat method as previously described (14). For some assays, 500 ng of purified, recombinant human colipase was added (15). To determine colipase activity, a portion of the extract was heated for 15 min at 65C to inactivate endogenous lipases. Both lipase and colipase activity were determined using 1 mg of protein from the heat-inactivated extract in the standard pH-stat method (14). All assays were done in duplicate. Another 100 until the sample was nearly dry, < 10 test. Comparisons of multiple means were done by one-way ANOVA with Tukeys test. < 0.05 was considered significant and was 0.05 for both tests. All values are means SD. RESULTS To determine whether the 4-d-old pancreas contains a colipase-dependent lipase activity, we made extracts of the pancreata from 4-d-old colipase-deficient pups and determined lipase activity. In the absence of added colipase, the extract had an activity of 1450 125 mU/mg protein. The addition of exogenous colipase significantly increased the activity to 3010 243 mU/mg protein (< 0.001 vs. no colipase by test, Pamiparib = 4). After incubation of the extract with an anti-human pancreatic triglyceride lipase polyclonal antibody that cross-reacts with PLRP2 and another homologue, PLRP1, no activity was detected (= 3) (9). Similar activities were found in colipase-depleted extracts from the pancreata of 4-d-old wild-type littermates of colipase-deficient pups. The activity in the absence of colipase was 1510 103 mU/mg protein. Adding colipase to the assay increased the activity to 3260 387 mU/mg protein (< 0.002 vs. no colipase by test, = 3), and preincubation of the extract with the anti-human pancreatic triglyceride lipase polyclonal antibody abolished activity. The wild-type values and the corresponding Pamiparib values from the colipase-deficient pups did not differ. We next measured lipase activity in colipase-depleted pancreatic extracts from 4-d-old PLRP2-deficient pups. The extract contained 630 50 mU/mg protein of lipase activity (= 4). This activity was significantly decreased compared with the activity in extracts of procolipase-deficient and wild-type mice (< 0.001 for both comparisons by one-way ANOVA with Tukeys test). Adding exogenous colipase to the assay did not increase the activity, which was 620 76 mU/mg protein (= 4). Preincubation with either anti-pancreatic triglyceride lipase antibody or anti-carboxyl ester lipase antibody did not inhibit the activity, 640 63 and 630 71 mU/mg protein, respectively (= 3). To help identify the source of lipase activity in the extracts from the pancreas of 4-d-old colipase-deficient pups, we took advantage of the tight association of PLRP2 with zymogen granule membranes and separated PLRP2 from soluble lipases (17). The starting extract contained 3300 276 mU/mg protein; 2740 253 mU/mg protein (83%).