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The nucleotide sequence of the pldA gene of Escherichia coli K-12, which codes for detergent-resistant phospholipase A (DR-phospholipase A), located in the outer membrane, was determined and the amino acid sequence of DR-phospholipase A was deduced. DR-phospholipase A contains 269 amino acids, resulting in a protein with a molecular weight of 30,809. It does not contain any cysteine residues and seems to be synthesized first as a precursor with a typical signal peptide composed of 20 amino acids. The NH2-terminus of the mature protein is glutamine, a polar amino acid, while other outer membrane proteins so far determined have a nonpolar amino acid there. The hydropathy profile of the deduced amino acid sequence of DR-phospholipase A was studied. Most of the region was rather hydrophilic and there were no stretches of hydrophobic amino acids. Computer analysis showed that there are no homologies between DR-phospholipase A and other extracellular phospholipases whose amino acid sequences are known. The candidates for the promoter region of the pldA gene, the 5'-flanking region, have a significantly high AT content, while the AT content of the coding region is about the same as the average AT content of the E. coli chromosome. A typical rho-independent transcription termination site is also present at the 3'-flanking region. This is the first example of the primary structure of a membrane-bound phospholipase.

Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.

An intracellular clotting factor, factor C, found in the horseshoe crab hemocytes is a lipopolysaccharide-sensitive serine-protease zymogen, which participates in the initiation of the hemolymph clotting system [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. The subsequent study of this zymogen, using various synthetic lipid A analogues, revealed that the zymogen factor C is rapidly activated by acylated (beta 1-6)-D-glucosamine disaccharide bisphosphate (synthetic Escherichia coli-type lipid A), and the corresponding 4'-monophosphate analogues. However, the corresponding non-phosphorylated lipid A did not activate factor C, indicating that a phosphate ester group linked with the (beta 1-6)-D-glucosamine disaccharide backbone is required for the zymogen activation. During these studies we also found that the zymogen factor C is significantly activated by acidic phospholipids, such as phosphatidylinositol, phosphatidylglycerol and cardiolipin, but not at all by neutral phospholipids. The rate of this activation, however, was affected markedly by ionic strength in the reaction mixture, although such an effect was not observed in the lipid-A-mediated activation of factor C. A variety of negatively charged surfaces, such as sulfatide, dextran sulfate and ellagic acid, which are known as typical initiators for activation of the mammalian intrinsic clotting system, did not show any effect on the zymogen factor C activation. These results suggest that lipid A is the most effective trigger to initiate the activation of the horseshoe crab hemolymph clotting system.

The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.

Detergent-resistant phospholipase A (DR-phospholipase A) of E. coli is a 28K-dalton protein and is exclusively located in the outer membrane. We cloned the pldA gene of E. coli, which is responsible for the activity of DR-phospholipase A. Strains bearing the plasmid which contained the pldA gene yielded a large amount of the outer membrane protein with a molecular weight of about 28K daltons and overproduced 20 to 65 times as much DR-phospholipase A activity as the wild type strain. Experiments with minicells and maxicells revealed that the pldA-containing plasmid was coding for a 28K protein. These results strongly indicated that pldA is the structural gene for DR-phospholipase A. There was apparently no difference with respect to the association of the enzyme with the envelope fraction between the overproducer and the wild type strain. The overproduced enzyme was properly transported to the outer membrane. Neither the growth rate nor the phospholipid composition of the overproducer was remarkably different from in the wild type strain. Thus, the overproduction of DR-phospholipase A apparently caused no phenotypic variations. E. coli has very excessive ability to transport and integrate the outer membrane protein.

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.

Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.

Two kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.

The incorporation of 2-acyl lysophospholipids into Escherichia coli , and their metabolism were studied. 2-[ 14 C]Acyl lysophosphatidylethanolamine could penetrate into E. coli cells and was mainly incorporated into phosphatidylethanolamine. 2-Acyl lysophosphatidylethanolamine was partially degraded, but some of it was incorporated into membrane phospholipids by acylation. 2-Acyl lysophosphatidylcholine also entered cells and was acylated to phosphatidylcholine. The acylation of 2-acyl lysophospholipid by the envelope fraction was also studied. Fatty acids were incorporated into 2-acyl lysophospholipids by the envelope fraction in the presence of ATP and Mg 2+ , and the incorporation was stimulated by acyl carrier protein, but not by coenzyme A. No acylation was observed with acyl coenzyme A as acyl donor. The acylation activities of the inner and outer membranes were examined. Pathways for degradation and modification of membrane phospholipids in E. coli are proposed.

The genes coding for the phospholipid degradation enzymes in E. coli, detergent-resistant (DR-) phospholipase A (pldA) and lysophospholipase L2 (pldB), were cloned together on the plasmid pKO1 (Homma, H., Kobayashi, T., Ito, Y., Kudo, I., Inoue, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1983) J. Biochem. 94, 2079-2081). To study their gene organization, a transducing lambda phage, lambda pldApldB, carrying both the pldA and pldB genes was constructed in vitro from plasmid pKO1. Viable deletion mutants of lambda pldApldB were isolated by EDTA killing, and their deleted DNA regions were determined by electron microscopic analysis of appropriate heteroduplexes. The activities of DR-phospholipase A and lysophospholipase L2 were also measured in lysates of cells infected with the deletion phages. The DNA region essential for the expression of each lipolytic activity was determined. In addition, proteins coded by the bacterial DNA on the plasmids containing the pldApldB region to various extents were detected by the maxicell system. The results showed that the product of the pldB gene is a protein with molecular weight of 40,000. It was also shown that the pldB gene is located at a region about 3 kilobase from the pldA gene.

Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].

E. coli bearing hybrid plasmid pKOl (Oeda et al. (1981) Mol. Gen. Genet. 184, 191-199) expressed a large amount of lysophospholipase L2 activity. When a mutant which was defective in lysophospholipase L2 activity was transformed with plasmid pKOl, it overproduced lysophospholipase L2 activity. The gene responsible for the lysophospholipase L2 activity was designated as pld B. On the same hybrid plasmid another gene (pld A) coding for detergent-resistant phospholipase A (DR-phospholipase A) was also identified. These facts together with the results of a Pl transduction experiment revealed that the pld B gene must be between the pld A and met E genes on the E. coli chromosome.

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.