E. H. Cordes

Phospholipases: Melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes☆

Abstract Melittin isolated from the venom of the common honey bee is a potent activator for bee venom phospholipase A 2 -catalyzed hydrolysis of unsonicated liposomes of egg phosphatidyl choline. At 37 °C and pH 8, the rate of this enzymatic reaction is increased approximately 300-fold by the addition of 8 × 10 −5 m melittin. The magnitude of facilitation of the phospholipase A 2 reaction is much greater than that previously reported by other workers for systems involving sonicated egg phosphatidyl choline liposomes or Escherichia coli membrane fragments as substrates. Melittin having lysines quantitatively modified through reaction with methyl acetimidate is as effective a potentiator of phospholipase A 2 activity as the unmodified material. The same result was obtained for melittin in which the single tryptophan residue was modified. Melittin modified by succinylation retained approximately 50% of its capacity to facilitate phospholipase A 2 activity. In contrast, a modified melittin in which the C-terminal four amino residues were removed, acetimidated des(23–26)melittin, is a very poor activator, as is a mixture of this peptide with the C-terminal tetrapeptide. In contrast to the results with egg lecithin liposomes, melittin has little influence on the susceptibility of monomolecular aqueous solutions of dihexanoylphosphatidyl choline to phospholipase A 2 attack.

Kinetics of water penetration into unsonicated liposomes effects ofn-alkanols and cholesterol

SummaryThe rate of swelling of egg lecithin liposomes under osmotic shock has been studied employing a stopped-flow spectrophotometer. Incorporation of cholesterol and simple alcohols into the liposomal structure elicits a biphasic response in swelling rate: at low concentrations these additives increase but at high concentrations they decrease water permeabilty. For simplen-alkanols, the effects can be correlated with structure. Specifically, the concentration of alcohol required to elicit maximal permeability as well as the maximal permeability decreases with increasing length of the alcohol. These effects are accounted for on the basis of modification of the orientation and packing of lecithin molecules in the bilayer membrane of the liposome.

Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

SummaryThe rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C4 to C8. For longer alcohols (C9 to C12), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C4 to C8n-alkanols obey the relationshipp C=a logPoctanol+constant [cf. Hansch & Dunn,J. Pharm. Sci.61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

SummaryThe cortical tissue of rat brain was fractionated through zonal centrifugation in a continuous sucrose density gradient, yielding a variety of morphologically distinct membrane fragments derived from nerve-end particles possessing variable levels of activity of Na, K-dependent Mg-sensitive ATPase (Na, K-ATPase) and other enzymes. Upon addition of certain of the zonal fractions, particularly those rich in the ATPase and acetylcholinesterase activities, to one side of planar artificial membranes, formed from mixtures of oxidized cholesterol and alkanes and bathed in a solution containing sodium, potassium, and magnesium ions, direct current membrane resistance fell from one to three orders of magnitude. Subsequent addition of ATP to the same side of the membrane to which the ATPase was added (thecis side) led to the development of net short-circuit current flow and open-circuit potential across the membrane (thecis side being negative with respect to thetrans side). Development of the short-circuit current and open-circuit potential is dependent upon the presence of all the substrates of Na, K-ATPase as well as that of the enzyme itself. The net current flow is inhibited and the open-circuit potential discharged by the addition of ouabain to thetrans side of the membrane, of phospholipase A to thecis side, or of trypsin to either side of the membrane. These observations provide circumstantial evidence for the reconstitution of the active cation pump across the artificial bilayer. Efforts to effect a similar reconstitution across membranes of this and other compositions employing Na, K-ATPase preparations from beef heart, beef brain, cat brain, human red cells, rabbit kidney, and rat brain microsomes failed.

Natural abundance carbon-13 nuclear magnetic resonance spectra of the canine sciatic nerve.

The proton-decoupled natural abundance carbon-13 nuclear magnetic resonance spectrum of the canine sciatic nerve is virtually identical to that of canine adipose tissue and markedly similar to that of liquid triolein. No resonances assignable to cholesterol, glycolipids, or sphingolipids are detectable in the sciatic nerve spectrum despite their abundance in the myelin sheath of this nerve. However, many such resonances are observed in lipid extracts of the nerve. Chronmatographic analysis of specimens of canine and rabbit sciatic nerve has revealed that these contain sufficient triglyceride to account quantitatively for the observed spectrum. Proton nuclear magnetic resonance and spin-labeling results for preparations containing myelin, especially those derived from the peripheral nerve, should be critically examined for experimental artifacts reflecting the triglyceride content.

Phospholipases. II. Enzymatic hydrolysis of lecithin: Effects of structure, cholesterol content, and sonication

SummaryHydrolysis of unsonicated liposomes of egg lecithin catalyzed by several phospholipases is markedly activated by addition ofn-alkanols [Jain & Cordes,J. Membrane Biol.14:101 (1973)]. Further pursuit of these systems has established that several factors, including higher temperatures, increasing unsaturation of fatty acyl chains of the substrate, incorporation of cholesterol into the liposomes, and sonication, reduce the concentration ofn-hexanol required to elicit maximal activation for enzymatic hydrolysis. Moreover, sonication or incorporation of cholesterol into lecithin liposomes reduces from C8 to C7 and C6, respectively, the chain length of that alcohol eliciting maximal activation. These results are consistent with the hypothesis that sonication and increasing cholesterol content lead to liposomes which have a diminished thickness of the hydrocarbon region compared to that for unmodified liposomes derived from the same lecithin.

Incorporation of eel electroplax acetylcholinesterase into black lipid membranes. A possible model for the cholinergic receptor

Abstract Addition of low concentrations of acetylcholine or carbamylcholine to solutions bathing a black lipid membrane into which electroplax acetylcholinesterase has been incorporated elicits a dramatic increase in the membrane conductance. This change is prevented or reversed by addition of neostigmine or atropine to the system. The magnitude of the conductance increase of the acetylcholinesterase-treated membrane is proportional to the fourth power of the carbamylcholine concentration and, at constant carbamylcholine concentration, to the fourth power of the enzyme concentration in the medium.

Natural abundance carbon-13 nuclear magnetic resonance spectra of human serum lipoproteins.

Human serum lipoproteins have been studied by Fourier transform nuclear magnetic resonance of carbon-13 in natural abundance. Spectra of highdensity, low-density, and very-low-density lipoproteins were recorded and partly assigned. The prominent features of these spectra reflect the qualitative and quantitative composition of the lipid moiety of these complexes. The results suggest that carbon-13 nuclear magnetic resonance will be a useful technique for studies of the structural and dynamic parameters of lipoproteins.

Some properties of reactions catalyzed by pig brain NAD glycohydrolase.

Abstract Some aspects of pig brain NADase-catalyzed hydrolysis of NAD have been examined employing a partially purified enzyme and a titrimetric assay for enzyme activity. In accord with previous observations, this enzyme was observed to be catalytically active toward a number of NAD analogs and toward both NMN and nicotinamide nucleoside. It is subject to competitive inhibition by nicotinamide. Employing the natural substrate, the reaction velocity is observed to be rather insensitive to changes in pH, diminishing some fourfold as the pH is lowered from 9 to 4. The kinetic solvent deuterium isotope effect, V H 2 o V D 2 o , increases from 1.5 at pH 8.7 to 2.5 at pH 7.7. The pig brain NADase-catalyzed reaction is subject to marked inhibition by dithiothreitol resulting from rapid inactivation of the enzyme. Treatment of the inactivated enzyme with thiols has not resulted in reactivation of the enzyme.

Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes

SummaryApparent values ofKm and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.04mn-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C fromClostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values ofKm andVmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.