Studies on phospholipase A and its zymogen from porcine pancreas III. Action of the enzyme on short-chain lecithins

Abstract

1. 1. Short-chain lecithins (with C6, C7, and C8 fatty acid esters) have been used to study kinetically the enzymatic hydrolysis by pancreatic phospholipase A (EC 3.1.1.4) in aqueous systems, without the addition of emulsifiers.

2. 2. Although phospholipase A is able to attack these substrates in molecularly dispersed form, micellar solutions are hydrolyzed at a much higher rate.

3. 3. Of the three substrates examined, dioctanoyllecithin appeared to be the best substrate. Differences in maximal velocities might be interpreted in terms of interfacial area per molecule.

4. 4. Ca2+ is specifically required for activity of pancreatic phospholipase A. The kinetic results are consistent with a random mechanism in which the metal ion combines with the enzyme independently of the substrate. The substrate was found to combine with the enzyme independently of the metal ion concentration.

5. 5. Kinetic parameters were determined with diheptanoyllecithin as a substrate over a pH range from 5 to 9. Maximal binding of enzyme with substrate was observed at pH : 6. The affinity of the enzyme for Ca2+ decreased at pH values below 6.5.

6. 6. With diheptanoyllecithin as substrate, maximal velocities at infinite substrate and Ca2+ concentrations showed an optimum at pH 5.75.

7. 7. NaCl at high concentrations (up to 3.9 M) gave a 80-fold stimulation of the vmax (diheptanoyllecithin as substrate). The Ks value decreased slightly with increasing salt concentrations, while the KCa2+ increased very strongly. The activating effect of salt is presumed to be caused by a change of the properties of the lipid-water interface.