Studies on the hydrolysis of lecithin by a Penicillium notatum phospholipase B preparation

Abstract

Preparations of the phospholipase B of Penicillium notatum can hydrolyze lecithin to glycerlphosphorylcholine and free fatty acids, if certain activating lipids are added to the system. Cardiolipin, liver polyglycerolphospholipid and monophosphoinositide were the most effective activating lipids; tripalmitin and tristearin were less effective, and certain fatty acids showed very low activity. All other lipids and non-lipid substances tried were ineffective. A certain threshold concentration of activating lipid was necessary before lecithin hydrolysis began. However, lecithin attack occurred if 2 activating lipids were added at just below their individual threshold concentrations. Optimum lecithinase activity occurred at pH 3.1-3.4 when either cardiolipin or monophosphoinositide was used as the activating lipid, and at pH 4.2 when tripalmitin was used to promote lecithin hydrolysis. The promoted lecithinase activity was strongly inhibited by bivalent-metal ions such as Ca++ or Mg++ (1.67 mM). These did not inhibit lysolecithin hydrolysis by the enzyme preparation. Fluoride completely inhibited promoted lecithinase activity and lysolecithin hydrolysis at 4.1 mM and inhibition was still apparent at 0.41 mM. Splitting of the acyl fatty-ester bonds in the activating lipids cardiolipin or monophosphoinositide by mild alkaline hydrolysis resulted in a complete loss of the ability to promote lecithin hydrolysis. Monophosphoinositide (but not cardiolipin or tripalmitin) was hydrolyzed by the enzyme preparation and this attack was inhibited by lecithin. No transfer of fatty acids from lecithin labeled with C14-fatty acid to monophosphoinositide could be demonstrated in the presence of the enzyme preparation. The activating lipids caused no detectable precipitation of phospholipase B on the surface of the lecithin particles, as measured by the lysolecithinase activity of the particles and supernatant. Small quantities of cardiolipin and monophosphoinositide that promoted lecithinase activity completely changed the surface character of lecithin particles in aqueous emulsion, so that the lecithin became no longer extractable into organic solvents such as ether. Both the surface change and the enzymic activity could be prevented by the addition of Ca++ ions. It is suggested that cardiolipin and monophosphoinositide promote lecithin hydrolysis by P. notatum phos-pholipase B preparations by introducing on the surface of the lecithin particles, certain polar groups which are necessary for the enzymic attack.