Inhibiting the Activity of Calf Pregastric Lipase: Effect of Bile Salts, Lecithin, Liposomes, Phenyl Boronic Acid and Diethyl 4-Nitrophenyl Phosphate

Abstract

A commercial extract from oropharyngeal tissue of calves has been used as the source of partially purified pregastric lipase. Activity of the enzyme against 4-nitrophenyldecanoate was inhibited by the conjugated bile salts taurocholate, taurodeoxycholate, and tauro- and glyco-chenodeoxycholate in their monomeric form. Although solutions of L-α-lecithin (0-0.75 mg mL -1 ) enhanced the activity of the lipase at all concentrations studied, with maximum rate enhancement (∼190%) occurring within the range (0.11-0.34) mg mL -1 , even this concentration of L-α-lecithin could not remove the inhibitory effect of the bile salts. The Michaelis-Menten parameters, V and K M , were determined for the activity of the enzyme against 4-nitrophenylacetate in the absence and presence of egg phosphatidylcholine (egg-PC) and egg-PC:cholesterol (10:1 mol/mol) liposomes. While values of V decreased slightly (and to the same extent) in the liposomal suspensions, the value of K M was decreased by 50% in the normal liposome but increased by 30% in the cholesterol impregnated liposome. The phenyl boronic acid / lipase dissociation constant was evaluated as 1.9 mM and pronounced inhibition was obtained in the presence of diethyl 4-nitrophenyl phosphate (E600). These inhibition results confirmed the presence of serine in the active site of calf pregastric lipase.