P.P.M. Bonsen

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.

Studies on phospholipase a and its zymogen from porcine pancreas IV. The influence of chemical modification of the lecithin structure on substrate properties

Abstract 1. 1. A series of chemically modified lecithins were used to investigate by kinetic analyses their substrate c.q. inhibitor properties for porcine pancreatic phospholipase A. The substrate analogues used were systematically modified in: the stereochemical configuration, the susceptible ester bond, the phosphate moiety, the alkylchains, the glycerol backbone and in the position of the phosphorylcholine moiety. 2. 2. The desired relationship between chemical structure and inhibitory properties requires elimination of purely physical effects of the inhibitor on the organization of the substrate molecules at the lipid-water interface. 3. 3. Lecithins of the opposite stereochemical configuration and certain lecithin analogues with a modification of the susceptible ester bond were found to be purely competitive inhibitors. The 1-sn-phosphatidylcholines have Ki values identical to the K8 values of the corresponding 3-sn-phosphatidylcholines. The lecithin analogues with an acylamide linkage at the 2-position were found to be the most potent competitive inhibitors, while on the contrary substitution of the acylester bond by a sulfonyl ester linkage does not give rise to inhibitory properties. 4. 4. Lecithins with a modification in the glycerol-phosphate bond and in the position of the phosphorylcholine moiety are substrates, but exhibit much lower V values and their binding constants are similar to those of the corresponding normal substrates. 5. 5. Introduction of two methyl groups at the carbon atom adjacent to the carboxyl in the acyl chain of the potentially susceptible ester bond gives a lecithin which is not degraded by the enzyme. The presence of only one methyl branch in this position greatly diminishes the hydrolysis rate, probably due to steric hindrance. 6. 6. Increasing the distance between the susceptible ester bond and the phosphate moiety in a lecithin by introducing a methylene group completely abolishes enzymatic activity. These lecithin analogues were found to be competitive inhibitors. 7. 7. The minimal requirements for a phospholipid to be a substrate for phospholipase A, as established earlier, should be extended to include the fact that the phosphate moiety can be replaced by a phosphonate or sulfonate group.

Hydrolysis of phosphoglycerides by purified lipase preparations I. Substrate-, positional- and stereo-specificity

Abstract 1. 1.|Purified lipase preparations (EC 3.1.1.3) from porcine pancreas and from the mold Rhizopus arrhizus hydrolyze exclusively the fatty acid ester bond at the 1-position of all common types of phosphoglycerides, regardless of the nature and distribution of the fatty acid constituents. 2. 2.|Both enantiomeric forms of phosphatidylcholine are hydrolyzed at a similar rate by these enzymes, indicating that the latter lack stereo-specificity. 3. 3.|The susceptibility of several synthetic analogues of choline phosphoglycerides, modified in the nature and type of bond at the 1- and 2-positions, as well as of phosphotriester derivatives to lipase was compared to that of phosphatidylcholine. It could be tentatively concluded that the susceptibility of the 1-acyl ester bond to lipase is influenced by the type of bond present at the 2-position.

Studies on phospholipase A and its zymogen from porcine pancreas: I. The complete amino acid sequence

Abstract Phospholipase A from porcine pancreas was subjected to digestion with trypsin after sulfitolysis. The resulting peptides were purified by a combination of Sephadex filtration, electrophoresis and chromatography on paper. The amino acid sequence of these peptides was determined by Edman degradation and, occasionally, hydrolysis with carboxypeptidases A and B. Alignment of the tryptic peptides into a single chain containing 123 amino acids was determined from larger overlap peptides. Some of these peptides were obtained from tryptic digests of sulfitolyzed phospholipase A after amidination of the lysine residues. Additional peptides, utilized for the correct positioning of the tryptic peptides, were obtained from cyanogen bromide fragments before and after chymotryptic digestion. Finally, confirmatory evidence for the proposed arrangement was provided by digestion of the sulfitolyzed enzyme with thermolysin. These results, along with the known sequence of the activation peptide, attached at the N-terminal end of phospholipase A, also provide the amino acid sequence of the zymogen.

Studies on cardiolipin III. Structural identity of ox-heart cardiolipin and synthetic diphosphatidyl glycerol

Chemical synthesis of diphosphatidyl glycerol, a long-chain fatty acid ester of diphosphatidyl glycerol, phosphatidyl diglyceride and phosphatidyl glycerophosphate has stimulated a structural comparison with natural cardiolipin. Although in certain properties the various polyglycerol phospholipids are quite similar, the results of enzymic hydrolyses with phospholipase A (EC 3.1.1.4), acylation studies, optical rotation measurements and chromatography of the intact phospholipids and deacylated products indicated that beef-heart cardiolipin has a diphosphatidyl glycerol structure. Conclusive evidence was obtained by means of the breakdown of the phospholipids with phospholipase C (EC 3.1.4.3). The enzyme was found to hydrolyse both natural and synthetic diphosphatidyl glycerol into 1,2-diglyceride and 1,3-glycerol diphosphate, phosphatidyl glycerophosphate being an intermediate hydrolysis product.

CHEMICAL SYNTHESIS OF SOME LECITHIN ANALOGUES POTENTIAL INHIBITORS OF PHOSPHOLIPASE A

The chemical synthesis of a number of lecithin analogues is described. These compounds were prepared to study inhibitory properties in the enzymatic hydrolysis of normal lecithins by porcine pancreatic phospholipase A. The structures of the synthesized products resemble very much those of the normal lecithins, but differ in most cases in that part of the molecule which is supposed to be important either for the binding with phospholipase A or for the catalytic reaction. The synthesized compounds include stereo- and structural isomers of the normal lecithins (= substrates for phospholipase A) and lecithins modified in the alkyl chain, the ester bond, the glycerol backbone, and the phosphate moiety.

Studies on phospholipase A and its zymogen from porcine pancreas: II. The assignment of the position of the six disulfide bridges

Abstract Porcine pancreatic phospholipase A and its zymogen are single-chain proteins consisting of 123 and 130 amino acids, respectively. Both proteins contain twelve half-cystine residues and the absence of free sulfhydryl groups indicates the presence of six disulfide bridges. To assign the positions of these cystine bonds in the protein, enzymatic digestion was performed with pepsin, chymotrypsin and thermolysin under conditions which minimize disulfide interchange. After isolation of the cystine-containing peptides, the positions of the six disulfide bonds were established.

Structural investigations on glucosaminyl phosphatidylglycerol from Bacillus megaterium

Abstract 1. 1. Glucosaminyl phosphatidylglycerol from Bacillus megaterium was converted into phosphatidylglycerol and 2,5-anhydromannose. The stereochemical configuration of phosphatidylglycerol was investigated with phospholipase A, phospholipase C, and glycero-3-phosphate dehydrogenase, and was found to be 1,2-diacyl-sn-glycero-3-phosphoryl-1′-sn-glycerol. 2. 2. Partial acid hydrolysis of the phospholipid yielded glucosaminylglycerol, which was shown to be identical with 2′-O-(2-amino-2-deoxy-β-glucopyranosyl)-glycerol. 3. 3. 1′-O-(2- Amino -2- deoxy -β- d - glucopyranosyl ) glycerol , 2′-O-(2- amino -2- deoxy -β- d - glucopyranosyl ) glycerol and 2′-O-(2- amino -2- deoxy -β- d - glucopyranosyl )-1′- phosphorylglycerol were synthesized and compared with glucosaminylglycerol and glucosaminyl glycerophosphate derived from the phospholipid. 4. 4. The structure of the phospholipid was established as 1,2- diacyl -sn- glycero -3- phosphoryl -1′[2′-O-(2″- amino -2″- deoxy - d - glucopyranosyl )]-sn- glycerol .

Preparation of immunologically responsive liposomes with phosphonyl and phosphinyl analogs of lecithin.

Abstract Liposomes, which release trapped glucose marker upon incubation with an appropriate antiserum and complement source, have been prepared from analogs of lecithin that cannot serve as substrates for phospholipases A, B, C, or D. These results confirm and extend previous studies with radioactive liposomes which have suggested that direct attack on membrane phospholipids by the above enzymatic activities may not be responsible for complement-dependent damage.

Silica gel stimulates the hydrolysis of lecithin by phospholipase A

Abstract 1. 1. Silica gel stimulates the hydrolysis of aqueous lecithin suspensions by phospholipase A. The activation is slightly greater than that caused by ether and takes place equally well in bulk suspensions of silica gel or on thin-layer Chromatographic plates prepared with silica. 2. 2. The hydrolyses of lecithin by phospholipase C and of triolein by lipase are not affected by silica under the reaction conditions employed. 3. 3. In light of these findings, it is advisable to employ an independent means of stopping phospholipase A reactions prior to product separation in chromatographic systems.