Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.     

Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.     

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.     

Properties of phospholipase A2 isolated from rat serum

Phospholipase A2 was extensively purified (1300- to 1400-fold) from rat serum using Sephadex G-100 chromatography. It eluted at a position corresponding to a molecular mass of about 15 kDa. This one purification step gave two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster component had a molecular mass of 16 kDa and the slower band likely contained an aggregate of the faster component. Activity was associated with protein bands on nondenaturing gels. Enzyme activity was assessed using phosphatidylcholine or phosphatidylethanolamine labelled at sn position 2 with radioactive arachidonate. Phosphatidylethanolamine gave higher specific activities than phosphatidylcholine. The enzyme has an absolute requirement for Ca2+ and a pH optimum at 7.4. This pH optimum was more prominent for phosphatidylethanolamine. Activity was inhibited by oleate or arachidonate when phosphatidylcholine was used as substrate, but added free fatty acid did not significantly affect the hydrolysis of phosphatidylethanolamine. Addition of bovine serum albumin (fatty acid free) to assays increased the rate of release of arachidonate from phosphatidylcholine, but not from phosphatidylethanolamine. Phospholipase A2 is present in serum likely as a consequence of blood coagulation and may release fatty acids from cellular membranes following hemorrhage.     

Isolation and characterization of a new lectin from plasma of fish Channa punctatus

A soluble lectin is purified to apparent homogeneity from plasma of Channa punctatus by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose. The lectin has 140 kDa native molecular mass and 68 kDa subunit molecular mass, as determined by native and sodium dodecyl sulphate denaturing polyacrylamide gel electrophoresis, respectively. The lectin agglutinates human A and AB blood groups and rat, mice and guinea pig erythrocytes in the presence of Ca2+ and Mg2+ or Mn2+ ions. These divalent cations, but not thiol group, are obligatory requirements for the lectin activity. Gal(beta 1----3)GalNAc (0.09 mM) is the most potent inhibitor of the lectin.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.     

A cytosolic protein activator of cardiac sarcolemmal phosphoinositide phospholipase C

Ca2+ dependent polyphosphoinositide phospholipase C (PLC) activity in cardiac sarcolemma hydrolyzed both endogenous and exogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with an associated increase in inositol bisphosphate (IP2). Dialyzed cytosol and certain fractions of cytosol isolated by anion exchange or gel filtration chromatography activated sarcolemmal PLC activity by approx. 100%. The PLC activator eluted with an apparent molecular weight of 160 Kdal on a Sephacryl 300 column and was destroyed by heat or trypsin treatment. Exogenous 3H-PIP2 was not hydrolyzed by cytosolic fractions containing sarcolemmal PLC activator. These studies demonstrate that the polyphosphoinositide PLC in cardiac sarcolemma is regulated by a cytosolic protein.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Identification of a calcium-independent phospholipase A2 in rat lung cytosol and differentiation from acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether)

The 100 000 X g supernatant of total rat lung homogenate was found to contain at least three phospholipase A2-type activities. Gel filtration separated a low molecular weight and Ca2+-requiring phospholipase A2 from Ca2+-independent acylhydrolase peak with an apparent higher molecular weight. Upon DEAE-cellulose chromatography this fraction was separated into a Ca2+-independent acylhydrolase and a Ca2+-independent platelet-activating factor-acetylhydrolase with no apparent overlap in acyl chain length specificity. The long-chain acylhydrolase was shown to exhibit specificity for the ester bond at the sn-2-position. Ca2+-independent phospholipase A2 activity was inhibited by p-bromophenacylbromide and was resistant to diisopropylfluorophosphate. In contrast, the Ca2+-independent acetylhydrolase activity was inhibited by diisopropylfluorophosphate but was unaffected by p-bromophenacylbromide.     

Purification of phospholipase D from citrus callus tissue

Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg²⁺-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg²⁺-independent.     

Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions

Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLA2 activities were demonstrated: PLA2-H (200-500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1-100 microM Ca2+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2(+)-dependent manner. The association was completed within 5-10 min at 25 degrees C both at 10 microM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLA2, are present.     

Purification and characterization of phospholipase B from Kluyveromyces lactis, and cloning of phospholipase B gene

Phospholipase B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid substrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed substrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+. cDNA sequence of PLB (KlPLB) was analyzed by a combination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delbrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused loss of the enzymatic activity at both pH. These results suggested that PLB activity are dependent on a catalytic mechanism similar to that of cytosolic phospholipase A2.     

Purification and partial characterization of protein phosphatases from rat thymus

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.     

Solubilization and partial characterization of phospholipase A from rat heart sarcoplasmic reticulum

Phospholipase A has been solubilized from the sarcoplasmic reticulum of rat heart by treatment with Tris buffer, potassium chloride, taurodeoxycholate or octyl glucoside. On HPLC gel permeation, two phospholipases were identified at the void volume of a TSK 3000 column and at an apparent molecular mass of 60 kDa. The two activity peaks exhibited a predominance of phospholipase A1 activity (83-91%) and a lesser phospholipase C activity (4-9%) using sonicated 1-palmitoyl-2[1-14C]oleoylphosphatidylcholine liposomes as substrate. The voiding phospholipase A peak, which represented the bulk of the recovered activity, exhibited a requirement for calcium ions in the 0.3-3 microM range. The heat stability and response to mercuric ions was studied and some similarities were noted between the solubilized sarcoplasmic reticulum phospholipases A and the cytosolic phospholipases A of rat heart. It is speculated that the cytosolic phospholipase A which we reported earlier may represent in part phospholipase A released from sarcoplasmic reticulum during isolation of the subcellular membrane fractions.     

Dynamic interaction between components of hexaprenyl diphosphate synthase from Micrococcus luteus BP-26

Hexaprenyl diphosphate synthase from Micrococcus luteus BP-26, which has been known to be dissociated into two essential components, designated as components A and B, during hydroxyapatite chromatography [Fujii, H., Koyama, T., & Ogura, K. (1982) J. Biol. Chem. 257, 14610-14612], was also resolved similarly by Sephadex G-100 or DEAE ion-exchange chromatography. Each component takes various self-aggregated forms. The apparent molecular mass of component B estimated by gel filtration on Superose 12 varied depending on its concentration, ranging from approximately 18 to 49 kilodaltons (kDa). On the other hand, the apparent molecular mass of component A varied depending on not only its concentration but also the ionic strength of the medium, ranging from approximately 13 to 24 kDa. When a mixture of components A and B preincubated in the presence of Mg2+ but in the absence of substrate was subjected to Superose 12 gel filtration, they were eluted at positions identical with those observed when they were chromatographed individually. In contrast, when a mixture of components A and B incubated in the presence of Mg2+ and substrates was filtrated on Superose 12, the elution positions were markedly changed, showing that an approximately 24-kDa aggregate of component A (designated as A) and an approximately 27-kDa aggregate of component B (designated as B) were the major species. Evidence was also obtained to show that farnesyl diphosphate (FPP) binds to the components to form an aggregate, A-B-FPP-Mg2+, which probably represents an intermediary state of enzyme catalysis.     

巴西橡胶树胶乳磷脂酶A_2的分离纯化及部分酶学性质鉴定

通过丙酮沉淀、DEAE-纤维素离子交换柱层析和Sephadex G-100凝胶过滤柱层析等分离纯化技术,对巴西橡胶树胶乳C-乳清磷脂酶A2进行分离纯化。用SDS-PAGE测定其亚基的相对分子量。测定该酶最适温度和pH,动力学常数Km和Vmax。并测定Ca2+和La3+对酶活性的影响。结果显示:该酶被纯化了49.47倍,产率为5.12%。SDS-PAGE检测为单一条带,其亚基相对分子量约43kDa。最适反应温度为37℃,最适反应pH为8.0,Km为0.44mmol·L-1,Vmax为7.22μmol.(mL.min)-1。最适Ca2+浓度为50μmol·L-1,稀土元素La3+离子对磷脂酶A2活性有抑制作用,但加入Ca2+后可缓解La3+对磷脂酶A2活性的抑制作用。胶乳C-乳清磷脂酶A2与其他植物磷脂酶A2在Ca2+的依赖性上存在差异。研究结果为今后探索橡胶树胶乳磷脂酶A2的催化机理、调节机理及生理功能等奠定了基础。