Arend J. Slotboom

The effect of the polar headgroup on the lipid-cholesterol interaction: a monolayer and differential scanning calorimetry study.

Since the sterol 3β-OH group is essential for the lipid-sterol interaction, the interaction of cholesterol with natural phospholipids and glycolipids and synthetic phospholipids and analogs, differing in the polar moiety was studied in monolayers and liposomes. In monolayers, the interaction was measured as a reduction in the mean molecular area (condensing effect). In liposomes by differential scanning calorimetry as a reduction in the energy content of the crystalline → liquid-crystalline phase transition (liquefying effect). The presence of the oxygen atoms of the acyl ester linkages and of the oxygen atoms connecting phosphorus and carbon are not essential for the lipid-sterol interaction as determined by the above methods. Measurements with monoglucosyldiglyceride and diglucosyldiglyceride even reveal that the phosphorus and choline moiety are not required for the interaction. This indicates that it is unlikely that a specific binding of the sterol-OH group with any polar part of the lipid molecule is essential for the condensing or liquefying effect. The need for a 3β-OH group can neither be explained by a cooperative effect of the C19 methyl group and the 3β-OH group in the lipid-sterol interaction since 19-norcholesterol shows the same effect as cholesterol. The effect of cholesterol upon the crystalline → liquid-crystalline phase transitions in a codispersion of (1,2-dioleoyl)lecithin and (1,2-distearoyl)lecithin was studied by differential scanning calorimetry. At low concentrations (less than 25 mole%) cholesterol preferentially associates with (1,2-dioleoyl)lecithin. At higher concentrations cholesterol interacts with (1,2-distearoyl)lecithin as well. This indicates that when cholesterol is present in membrane with lipids in both the crystalline and liquid-crystalline state, cholesterol preferentially interacts with these lipids which are in the liquid-crystalline state.

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.

On the specificity of rat-liver lysophospholipase

Abstract 1. 1. A study on the specificity of rat-liver lysophospholipase activity (EC 3.1.1.5) revealed that both 1-acyl-sn-glycero-3-phosphorylcholine and 2-acyl-sn-glycero-3-phosphorylcholine are deacylated. From both positional isomers the unsaturated analogs appeared to be degraded at higher rates. 2. 2. Circumstantial evidence is presented indicating that 2-acyl-sn-glycero-3-phosphorylcholine can be attacked directly by this lysophospholipase activity without a prior migration of the fatty acyl constituent. 3. 3. Compounds lacking the free hydroxyl group present in lysophosphatidyl-cholines, e.g. acyl-ethylene glycolphosphorylcholine and 1-acyl-propane diol-3-phosphorylcholine, also fall in the enzymes range of specificity. 4. 4. Mono-acyl derivatives of sn-glycero-1-phosphorylcholine, sn-glycero-2-phosphorylcholine, as well as sn-glycero-3-phosphorylcholine, were found to be degraded. 5. 5. Inhibition of lysophospholipase activity by various agents exhibited the same effect on the deacylation of both 1-acyl- and 2-acyl-sn-glycero-3-phosphoryl-choline. 6. 6. The degradation of mono-acyl-phosphatidylcholine appeared to be strongly inhibited in the presence of phosphatidylcholine.

The acylation of isomeric monoacyl phosphatidylcholines

Abstract 1. 1. A comparison has been made of 1-acyl-3-sn-phosphatidylcholine and 2-acyl-3-sn-phosphatidylcholine as acyl acceptors in the synthesis of phosphatidylcholine as catalysed by acyl-CoA: monoacylphosphatidylcholine acyltransferase. With 1-acyl-3-sn-GPC, the following pattern for the extent of acyl transfer was observed: oleate > linoleate > laurate > palmitate ⩾ stearate. With 2-acyl-3-sn-GPC as acceptor, this sequence was: stearate > palmitate > laurate ⩾ oleate > linoleate. 2. 2. The conversion of 1-[1-su14,C]palmitoyl-sn-glycero-3-phosphorylcholine and 2-[9,10−3H2]stearoyl-sn-glycero-3-phosphorylcholine into molecular species of 3-sn-phosphatidylcholine has been shown to be in accordance with the view that 1-acyl-3-sn-GPC is preferentially acylated with unsaturated acids, whereas 2-acyl-3-sn-GPC is better acylated with saturated acids. The labelled acyl constituents were recovered in the synthesized 3-sn-phosphatidylcholines at essentially the same positions as those at which they were originally located in the substrates. 3. 3. Monoacyl phosphatidylcholine, which was derived from sn-glycero-1-phosphoric acid, has been shown not to act as substrate for the acyl-CoA: monoacylphosphatidylcholine acyltransferases from rat liver microsomes.

Anchoring of phospholipase A2: the effect of anions and deuterated water, and the role of N-terminus region.

The effect of anions and deuterated water on the kinetics of action of pig pancreatic phospholipase A2 is examined to elaborate the role of ionic interactions in binding of the enzyme to the substrate interface. Anions and deuterated water have no significant effect on the hydrolysis of monomeric substrates. Hydrolysis of vesicles of DMPMe (ester) is completely inhibited in deuterated water. The shape of the reaction progress curve is altered in the presence of anions. The nature and magnitude of the effect of anions depends upon the nature of the substrate as well as of the anion. Substantial effects of anions on the reaction progress curve are observed even at concentrations below 0.1 M and the sequence of effectiveness for DMPMe vesicles is sulfate greater than chloride greater than thiocyanate. Apparently, anions in the aqueous phase bind to the enzyme, and thus compete with the anionic interface for binding to the enzyme. Binding of the enzyme to anionic groups on the interface results in activation and increased accessibility of the catalytic site possibly via hydrogen bonding network involving water molecule. In order to elaborate the role of the N-terminus region in interfacial anchoring, the action of several semisynthetic pancreatic phospholipase A2s is examined on vesicles of anionic and zwitterionic phospholipids. The first-order rate constant for the hydrolysis of DMPMe in the scooting mode by the various semisynthetic enzymes is in a narrow range: 0.7 +/- 0.15 per min for phospholipase A2 derived from pig pancreas and 0.8 +/- 0.4 per min for the enzymes derived from bovine pancreas. In all cases a maximum of about 4300 substrate molecules are hydrolyzed by each phospholipase A2 molecule. If anions are added at the end of the first-order reaction progress curve, a pseudo-zero-order reaction progress curve is observed due to an increased intervesicle exchange of the bound enzyme. These rates are found to be considerably different for different enzymes in which one or more amino acids in the N-terminus region have been substituted. Steady-state and fluorescence life-time data for these enzymes in water, 2H2O and in the presence of lipids is also reported. The kinetic and binding results are interpreted to suggest that the N-terminus region of phospholipase A2 along with some other cationic residues are involved in anchoring of phospholipase A2 to the interface, and the catalytically active enzyme in the interface is monomeric.

Effect of chemical modification on the activity of lipases in organic solvents

Lipases from Rhizomucor Miehei, Candida antarctica, and Fusarium solani pisi were chemically modified with the aim to improve their catalytic properties in organic solvents. The chemical modifiers, two activated polyethylene glycol derivatives and activated n-octanol, were covalently linked to lysine residues at the surface of the enzyme leading to varying surface hydrophobicities. The modified lipases were tested for hydrolytic activity in water and for transesterification activity in the organic solvents o-xylene, tert-butyl methyl ether, tert-butanol, and 2-butanone. Whereas the hydrolytic activity was only slightly affected by the modifications, the transesterification activities were influenced strongly even though the modified lipases were still not soluble in organic solvents. The most effective modifier is tryesyl-activated polyethylene glycol 2000 monomethyl ether, activating lipases up to 27-fold in organic solvents while it is the least hydrophobic. The more hydrophobic modifiers, tresyl-activated polyethylene glycol 400 mono-octyl ethyl (tOPEG) and tresyl-activated octanol (tOCT), may lead to inactivation. Co-lyophilization of unmodified Candida antartica lipase B (CALB) with additives such as polyethylene glycol dimethyl ether and crown ether also positively affects the activity of CALB in organic solvents. However, we found that covalent linking of MPEG to CALB is more effective because the activation by additives is partially lost during washing of the enzyme for reuse. The thermostability of CALB in o-xylene is not affected by modification, whereas in 2-butanone the thermostability is decreased by MPEG modification and increased by OPEG or OCT modification. Our results suggest that MPEG positively influences the porosity of the lipase aggregates in organic media, whereas OPEG and OCT induce tighter aggregates.

Simplified pathways for the preparation of some well-defined phosphoglycerides.

Abstract This paper describes a few pathways leading to the preparation of optically pure, mixed-acid 3- sn -, 2- sn -, and 1- sn -phosphatidylcholines. The application of both lipolytic enzymes and simple chemical transformations allow the conversion of these lecithins into various other well-defined phospholipids. The usefulness of the prepared phosphoglycerides in a number of biochemical problems is discussed.

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.

Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity.

Two anti‐inflammatory peptides corresponding to a high amino acid similarity region between lipocortins were synthesized and tested on their ability to inhibit porcine pancreatic phospholipase A2. Kinetic assays using monomeric and aggregated phospholipids did not reveal any phospholipase A2 inhibitory activity. The peptides did not inhibit phospholipase A2 activity on monolayers of negatively charged substrate and did not prevent phospholipase A2 action on mixed micelles of 1‐stearoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine and sodiumdeoxycholate. Ultraviolet difference spectroscopy did not show binding of the peptides to phospholipase A2. Therefore we conclude that these anti‐inflammatory peptides do not inhibit pancreatic phospholipase A2 in vitro, in contrast to the results recently published [(1988) Nature 335,726–730].