Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Activation of phospholipase A2 of human spermatozoa by proteases

The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A₂ activity (PA₂: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA₂ activity optimally with trypsin concentrations of 1.0–1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA₂ activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA₂ activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA₂ activation upon the addition of proteases, thus indicating that the increase in PA₂ activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A₂ that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A₂ could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.     

Immunocytochemical localization of phospholipase A2 in hamster spermatozoa.

Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.     

Immunocytochemical localization of phospholipase A2 in hamster spermatozoa

Summary Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte.     

The rapid purification and partial characterization of human serum angiotensinogen.

Human angiotensinogen has been purified 390-fold from serum by a rapid high-yielding procedure that involved chromatography on Blue Sepharose, phenyl-Sepharose, hydroxyapatite and immobilized 5-hydroxytryptamine (5-HT). Angiotensinogen was specifically bound to immobilized 5-HT, which effected a partial resolution into multiple forms, which were also evident when analysed by SDS/polyacrylamide-gel electrophoresis (Mr 59,400, 60,600, 62,600 and 63,800). This heterogeneity was confirmed by resolution into six main bands on isoelectric focusing, ranging from pI 4.40 to 4.82. N-terminal analysis, digestion with human renal renin and deglycosylation studies implied that the preparation comprised several forms of angiotensinogen, varying in their degree of glycosylation. The presence of sialic acid was shown to be a major factor in determining the heterogeneity.     

Characterization and partial purification of soluble, lysosomal phospholipase(s) A2 from adrenal medulla

Soluble, cation-dependent, lysosomal phospholipase A2 in bovine adrenal medulla has been biochemically characterized and partially purified, and its unique pH-dependent modulation by cations has been investigated. Chromatographically distinct activities with somewhat broad pI ranges centered at 7.8, 8.1, and 8.4 have been purified 83-, 1900- and 4400-fold, respectively, from the soluble fraction of tissue homogenates. With a specific activity of 4.2 mumol phospholipid hydrolyzed per mg protein per min, the fraction of pI 8.4 is the most highly purified lysosomal phospholipase A2 reported to date; yet silver staining of isoelectric focusing gels indicates that all three species are still only minor components of the protein mixtures with which they co-purify. Lysosomal phospholipase(s) A2 has an apparent molecular weight of 30,600, as determined by gel permeation chromatography; and is probably an oligomannose-containing glycoprotein as indicated by binding to concanavalin A-Sepharose and elution by methyl alpha-D-mannopyranoside. Cation concentrations modulate hydrolysis of biomembranous phospholipid, but not neat liposomal phospholipids, in a complex manner over a broad pH range (pH 4.0-8.0). Triton X-100 stabilizes the enzyme(s) but is inhibitory when present during assay; consequently, detergent-phospholipid mixed micelles are poor substrates. Thus, experimental results are dramatically dependent on the physicochemical nature of the substrate. The role of this phospholipase(s) A2 in the membrane fusion and lysis events of catecholamine secretion, as well as its regulation by cellular proteins, can now be investigated utilizing this partially purified enzyme(s).     

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.     

Calmodulin stimulates human platelet phospholipase A2.

Calmodulin is a ubiquitous Ca²⁺-binding protein, mediating the effect of Ca²⁺ on many enzyme systems and cellular reactions. Phospholipase A₂ (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca²⁺ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A₂ by Ca²⁺ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.     

Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.     

Partial purification and properties of phospholipase A2 from the starfish, Asterina pectinifera.

1. Phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) activity was shown to occur in the supernatant fraction of a freshly prepared homogenate from the pyloric caecum of starfish (Asterina pectinifera). 2. The phospholipase A2 has been isolated and purified 130-fold by ultracentrifugation, ammonium sulfate precipitation and column chromatographic procedures. 3. The purified enzyme was stable to heat at low pH values and the optimal pH was observed at approximately 9.0. 4. The enzyme activity was activated by Ca2+ and sodium deoxycholate, and was inhibited by EDTA.     

Characterization and partial purification of acid lipase from human leucocytes.

Hydrolysis of glycerol trioleate by human leucocytes was characterized and the enzymes responsible for this activity were obtained in a purified form by means of gel chromatography on Sephadex G-100 as well as by zonal ultracentrifugation followed by gel chromatography. The activity is localized in the granule fraction of leucocytes (15 000 X g, 20 min) and shows a sharp pH optimum at pH 5.25. As judged from the elution profile obtained by gel chromatography, two proteins are likely to contribute to the hydrolysis of glycerol trioleate. The approximate molecular weights of the two enzymes are 74 100 and 60 300, respectively. The activity is reduced in the presence of NaCl, KCl, CaCl2 as well as of p-hydroxymercuribenzoate. The enzymes are stable at -25 degrees C but loose about 50% of their activity within 48 h at 4 degrees C.