Abstract:
A commercially available pregastric lipase extract from calf has been partially purified by dialysis, anion-exchange chromatography and gel filtration. The protein fraction of interest, that with the greatest activity against tributyrylglycerol (TBG) was prepared as an emulsion with L-α-lecithin and casein and eluted off an anion-exchange column, after binding strongly to the column at lowered pH and subsequent removal with a high concentration of NaCl (l M). The enzyme thus obtained was named calf pregastric lipase (CPGL) and showed preferential activity against TBG (a short-chain homoacid triacylglycerol), with respect to high values of Vs in combination with diminished KM, in contrast to the values obtained with the water miscible ester, 4-nitrophenyl acetate (4-NPA). Values of VS, the limiting rate of hydrolysis at the lipid / water interface, for the CPGL catalysed hydrolysis of TBG and V, the limiting rate of hydrolysis in a homogeneous solution, for the CPGL catalysed hydrolysis of 4-NPA at pH 6.0 and 35 oc were 8.36 ± 0.44 U mg-1 and 3.60 ± 0.16 U mg-1, respectively. The respective values of KM under the same conditions of pH and temperature were 0.10 ± 0.02 mM for TBG as substrate, and 0.53 ± 0.09 mM for 4-NPA. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of the partially purified CPGL indicated the presence of three proteins with molecular weights (MW) corresponding to 44, 62 and 66 kDa. Comparison with literature MW values of pregastric lipases from other well-studied species (human, lamb, kid, goat and rat) identified the protein of interest at 44 KDa. This band was excised and subjected to five Edman degradation cycles. Although the MW of the partially purified CPGL is somewhat lower than the reported MWs of other ruminant PGLs, the resultant N-terminal sequence was in complete agreement with those previously reported for kid, goat, lamb and calf pregastric lipases (F, L, G, K, I). The lower MW lipase isolated in this study may have been partially deglycosylated or undergone uncontrolled proteolysis during the purification procedure.
The activity of partially purified CPGL against the short-chain homoacid triacylglycerol, TBG, was further studied over a range of pH values (5.5 to 7.5) and temperature conditions (30 ºC to 50 ºC) and, in the presence of possible inhibitors. The maximum rate of TBG hydrolysis catalysed by CPGL was at pH 6.5 and 40 ºC, and under these conditions the value of Vs, the limiting rate at the interface, was equal to 14.3 ± 0.7 U mg-1. The stoichiometry of the CPGL catalysed hydrolysis of TBG was determined as 'one to one', and CPGL was observed at this early stage of investigations to exhibit substrate specificity for TBG compared with the initial product of hydrolysis, 1,2-(2,3)-dibutyrylglycerol (DBG). The approximate rate ratio for the catalysed hydrolysis of TBG:DBG by CPGL was 30:1. Thermal inactivation of CPGL was evident at high temperatures and especially so in combination with high pH. The enzyme showed a degree of thermal resistance at the lower pH scale and also in the presence of the following proteins: bovine serum albumin, more casein, lactoglobulin and transferrin. The greatest stabilising effect was exerted by the milk proteins lactoglobulin and casein. CPGL was found to be relatively resistant to the gastric protease, pepsin, retaining approximately 70% activity in the presence of 2% w/v pepsin for 1 hour. Proteins added to the TBG emulsion decreased the observed activity of CPGL by either aligning at the lipid / water interface or acting as fatty acid receptors, or both. Short-chain fatty acids (4:0, 6:0 and 8:0), as opposed to the longer chain (10:0 and l2:0) fatty acids, in the TBG emulsion, seriously inhibited CPGL activity, suggesting that the hydrolysis of TBG is more dominant in the aqueous phase than at the interface.
The hydrolysis of 4-nitrophenyl acetate catalysed by CPGL was also examined over a range of pH values (6.0 to 8.0) and temperature (30 ºC and 50 ºC) values. Preliminary studies of the reaction milieu established that addition of up to 1.6% v/v acetonitrile, the solvent for the substrate, had very little effect on the measured pseudo first-order rate constant of hydrolysis. Michaelis-Menten parameters were determined in the presence of increasing concentrations of acetonitrile (0.66 - 6.6% v/v). As the concentration of acetonitrile increased, the limiting rate, V, decreased and the value of KM was unchanged. Hence the solvent behaved as a non-competitive inhibitor. Values of initial rate and Kψ were found to be buffer dependent, with relatively greater values being reported in bis tris buffer. Thus all studies were confined to the useful pH range of only one buffer, the choice of which was determined by the pH range under investigation. The maximum limiting rate, V, for the CPGL catalysed hydrolysis of 4-NPA occurred at pH ≤ 6.0 and across a broad temperature range of 35 ºC - 50 ºC. The onset of thermal inactivation of CPGL appeared under noticeably milder conditions than with TBG as substrate, due to less favourable binding of 4-NPA to CPGL. A pK2 value of 5.7 for the CPGL-4-NPA complex was obtained by fitting pH and Kψ data to the Dixon equation. Arrhenius plots of log Kψ against I/T for the CPGL catalysed hydrolysis of 4-NPA was linear up to 42 ºC allowing for calculation of the following thermodynamic parameters: Ea = 37 ± 2 kJ mol-1; A = 4600 ± 200 s-l; ∆H* = 35 ± 2 kJ mol-1; and ∆S* = -O.17 ± 0.02 kJ K-l mol-1.
Studies of increasing acyl chain length of 4-nitrophenyl esters and homoacid triacylglycerols showed that CPGL preferentially hydrolyses 4-nitrophenyl decanoate and TBG, respectively. Gas chromatography was used to analyse the hydrolytic products of bovine milkfat catalysed by CPGL. CPGL preferentially hydrolyses the short-chain fatty acids (C4:0 and C6:0) which are dominant at the sn-3 position of bovine milk. Thus, CPGL is both typoselective for short-chain fatty acids and stereoselective for the sn-3 position.
A study of the activity of CPGL in the presence of conjugated bile salts and egg phosphatidylcholine (PC) liposomes was undertaken to elucidate the viability of CPGL activity in the duodenum. All the conjugated bile salts studied, whether in the presence or absence of L-α-lecithin, diminished the initial rate of CPGL catalysed hydrolysis of the medium-chain ester, 4-nitrophenyl decanoate. With respect to the conjugated bile salt, the nature of the headgroup and the number and position of the hydroxyl substituents on the steroidal nucleus affect the degree of inhibition. The results indicated that CPGL is unlikely to contribute much, if at all, to fat digestion beyond the stomach (abomasum). Intercalation of 4-NPDe into egg PC liposomes and egg PC:cholesterol liposomes decreases the limiting rate of CPGL catalysed hydrolysis by effectively removing the substrate from CPGL. Phenyl boronic acid (BBA) and diethyl 4-nitrophenyl phosphate (E600) are known inhibitors of serine hydrolases. Both of these inhibited CPGL catalysed hydrolysis of 4-NPA. BBA inhibited CPGL activity in a competitive way and a plot of KM against BBA concentration gave a value of 1.9 mM for the competitive enzyme-inhibitor constant, Ki. This value is similar to that of KM in the absence of BBA and implies that BBA and 4-NPA have a similar affinity for CPGL. In the presence of E600, an irreversible serine specific inhibitor, the activity of CPGL decreased to l8% of the activity in the absence of E600, after 2-hours incubation. This suggests that an essential serine resides at the active site of CPGL.