G.H. de Haas

Purification and properties of phospholipase a from porcine pancreas

1. 1. Freshly prepared homogenates of pig pancreatic tissue contain a small amount of phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4); during autolysis, however, a considerable rise in lipolytic activity occurs. 2. 2. By use of heat treatment, (NH4)2SO4 precipitation and chromatographic procedures, the enzyme has been purified about 200 times and characterized by chemical and enzymatic procedures. 3. 3. The protein, which has a molecular weight of about 13 800 ± 500 appears to consist of one single polypeptide chain terminating in alanine (NH2) and cystine (COOH), and cross-linked intramolecularly by 7 disulphide bridges. 4. 4. The enzyme acts stereospecifically on all common types of 3-sn-phosphoglycerides, hydrolysing exclusively fatty acid ester bonds at the glycerol-C-2 position, regardless of chain length or degree of unsaturation. In contrast to the snake venom phospholipase A, the pancreatic enzyme shows a marked preference for anionic phospholipids such as phosphatidic acid, cardiolipin and phosphatidyl glycerol.

The substrate specificity of phospholipase A

Investigations on variously modified analogues of phospholipids elucidated the following substrate characteristics for phospholipase A (Crotalus adamanteus). 1. 1. Within the class of α-phosphoglycerides l-isomers are readily hydrolysed, while d-α-phospholipids appeared not to be attacked. 2. 2. l-α-Lecithins containing fatty acids with greatly varying chain length are susceptible to phospholipase A action; however, certain water-soluble short-chain compounds are hydrolysed at a very slow rate only. 3. 3. Aside from effects on the surface charge of the phosphoglyceride micelles the nature of the polar headgroup esterified to the phosphoryl moiety turned out not to form any prerequisite, and its presence even appeared to be dispensible. 4. 4. Contrary to a blocking of the amino function, protection of the hydroxyl function of phosphatidylethanolamine caused an inactivation of the substrate properties. 5. 5. Both, a γ-benzyl-β-acyl-α-phosphoglyceride and a β-acyl-lyso derivative, were hydrolysed, whereas the corresponding structural isomers carrying the fatty acid in γ-position appeared not to be susceptible to phospholipase A action. 6. 6. Glycol analogues were demonstrated to exhibit substrate activity. 7. 7. Phospholipase A catalyses the hydrolysis of a symmetric β-lecithin into an optical-active lysolecithin. 8. 8. An isolated specimen of cardiolipin was hydrolysed, whereas sphingomyelin resisted phospholipase A action.

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.

Purification from porcine pancreas of two molecular species with lipase activity

Abstract The two lipases existing in porcine pancreas and pancreatic juice have been fully purified by a method involving the six following steps: (1) almost complete delipidation of pancreas homogenates by solvent extraction; (2) (NH4)2SO4 fractionation; (3) removal of an acidic phosphatide by extraction and partition between n butanol and (NH4)2SO4; (4) chromatography on DEAE-cellulose (pH 9.0); (5) filtration through Sephadex G-100 and (6) separation of the two lipases by chromatography on CM-cellulose (pH 5.0). This method can be applied on a relatively large preparative scale. The two porcine lipases appear to be very closely related with regard to amino acid composition, molecular weight (45 000–50 000) and specific activity on long- or short-chain triglycerides.

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.

Purification and properties of an anionic zymogen of phospholipase a from porcine pancreas

Abstract 1. 1. This paper describes the isolation and purification of an enzymically inactive precursor of porcine pancreatic phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4). 2. 2. The protein, which has a molecular weight of about 15 000, appears to consist of a single polypeptide chain, terminating at the NH 2 region in the amino acid sequence: Glu-Gly-Glu-Ile-Ser-Ser-Arg-Ala......, and having cystine as COOH-terminal amino acid. 3. 3. The precursor molecule is activated by trypsin which splits the above -Arg-Ala-peptide bond, yielding active phospholipase A and the heptapeptide: Glu-Gly-Glu-Ile-Ser-Ser-Arg. 4. 4. In this released peptide, as well as in the precursor molecule itself, the N-terminal glutamic acid residue has no free α-NH 2 group. 5. 5. Phospholipase A, isolated from autolysed pancreatic tissue, appears to be identical with the product obtained by trypsin activation of the pure precursor.

The Synthesis of Phosphoglycerides and Some Biochemical Applications

Publisher Summary This chapter discusses the synthesis of phosphoglycerides and some biochemical applications. Lecithin, or phosphatidylcholine, is known to be widely distributed in nature. Although many, but not all, bacteria are devoid of lecithin and certain mammalian cells, this phospholipid is in general present in relatively high proportions. Many procedures have been developed for the isolation of lecithin from suitable sources, and the chromatographic methods particularly render preparations of excellent purity. Glycol analogs have often been synthesized to check synthetic methods prior to their application in the synthesis of the corresponding phosphoglycerides. These glycol derivatives, not detected in nature, are intermediate in their properties between diacyl phosphoglycerides and lyso compounds. A great advantage rendered by the utilization of synthetic phosphoglycerides in biophysical studies is the possibility of evaluating the effect of the nature of the acyl constituents on their behavior.

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.

Kinetic analysis of the hydrolysis of lecithin monolayers by phospholipase A

Enzymic hydrolysis by pancreatic phospholipase A (E.C. 3.1.1.4) of L-dioctanoyl-, L-didecanoyl- and L-didodecanoyllecithin monolayers was studied under constant surface pressure by measuring the amount of substrate which disappears per unit area per unit time. The reaction is first-order with respect to the total number of substrate molecules allowing the determination of a rate constant. Apparent limitations of the monolayer techniques are often caused by diffusion problems. Experimental conditions are discussed to detect and control these difficulties. pH dependence and calcium requirements of the enzymatic reaction are similar under monolayer and bulk conditions. For all three substrates plots of velocity vs. surface pressure show bell-shaped curves with a similar maximum rate at a surface pressure of about 8 dynes/cm. This result is discussed in relation to conformational changes in the lecithin molecules. With the monolayer techniques one can determine only a minimal specific activity, because of the unknown amount of enzyme involved in the catalysis. This minimal specific activity is compared with the value obtained with lecithin micelles as substrate in similar bulk conditions. Inhibition of the enzyme by competitive inhibitors present in mixed films cannot be studied by the monolayer technique. Comparison of the monolayer and bulk methods showed that both techniques are complementary.