Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.

Phospholipase C from Clostridium novyi type A. I.

1. Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography. 2. The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine. This preparation was free from protease, lipase and oxygen-labile delta-hemolysin. 3. Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates. 4. Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme. 5. This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine.

The presence of GPI-linked protein(s) in an archaeobacterium, Sulfolobus acidocaldarius, closely related to eukaryotes.

GPI-anchored proteins are distributed ubiquitously in eukaryotes, but not in procaryotes. By metabolic-labeling of Sulfolobus acidocaldarius cells, 14C-radiolabeled precursors of GPI and caldarchaetidylinositol were incorporated into 120, 143 and 185 kDa proteins. The 185 kDa protein was specifically solubilized by bacterial phosphatidylinositol-specific phospholipase C. Therefore, Sulfolobus proved to contain at least one GPI-anchored proteins.

Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.

A phosphatidylinositol-specific phospholipase C was purified from the culture broth of Bacillus thuringiensis to a homogeneous state as indicated by polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 312 units/mg, and the recovery of the enzyme activity was 27.2%. The purified enzyme (molecular weight: 23 000 +/- 1000) was maximally active at pH 7.5 and not influenced by EDTA. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and sphingomyelin. The products from phosphatidylinositol of enzyme reaction were diacylglycerol and myoinositol 1,2-cyclic phosphate. The enzyme activity was stimulated by sodium deoxycholate or Triton X-100. Divalent cations such as Ca2+, Mg2+ and Zn2+ were inhibitory at concentrations above 10(-3) M. KCl and NaCl were inhibitory at the concentration higher than 10(-2) M. Alkaline phosphatase, an ecto-enzyme located on the surface of plasma membrane, was released from the slices of rat liver, kidney, pancreas and intestine by the treatment with this phospholipase.

Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus.

A sphingomyelinase of Bacillus cereus was purified to a homogeneous state (512 U/mg, 2200-fold) as indicated by SDS-polyacrylamide gel electrophoresis and the molecular weight (23,300) was determined by sedimentation equilibrium. The enzyme contained loosely-bound magnesium atom. The addition of Mg2+ accelerated the enzyme reaction regardless of substrates and their physical state. The addition of Ca2+ also accelerated the enzyme reaction slightly, when water-soluble substrates, i.e., 2-hexadecanoylamino-4-nitrophenylphosphorylcholine and p-nitrophenylphosphorylcholine, were used as substrates. On the other hand, the addition of Ca2+ inhibited enzyme reaction when mixed micelles of either sphingomyelin and Triton X-100 or sodium deoxycholate were used. The surface charge on mixed micelles affected the enzyme reaction. When the mixed micelle of sphingomyelin and Triton X-100 was used as substrate, Ca2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 33 microM. On the other hand, when the mixed micelle of sphingomyelin and sodium deoxycholate was used as substrate, Ca2+ stimulated the enzyme reaction at lower concentration in the presence of a low concentration of Mg2+, although higher concentrations of Ca2+ were still inhibitory. In this case, added Ca2+ may be used as a substitute of Mg2+ to neutralize the negative charge on the mixed micelle, improving the accessibility of sphingomyelinase to the micellar substrate. A cationic detergent, cetyltrimethylammonium bromide, seemed to denature or inactivate the enzyme.

Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5'-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment.

In order to address the minimum domain of the COOH-terminal hydrophobic region responsible for GPI modification of bovine liver 5'-nucleotidase, we constructed a series of the deletion mutants of the COOH-terminus and expressed them in COS cells. Cells transfected by the deletion mutant of 6 amino acids (-IIILYQ) from the hydrophobic domain (-FSLIFLSVLAVIII-LYQ) did not show any elevation of cell surface-associated 5'-nucleotidase activity, whereas the 2 (-YQ) or 4 (-ILYQ) amino acid deletion mutant retained the bovine liver-derived activity on the cell surface as a GPI-anchored protein. Loss of half the hydrophobic domain (6 or 8 amino acids) resulted in accumulation of the activity in the cell. On the other hand, deletion of the whole hydrophobic domain (17 amino acids) or the entire cleaved-off domain (25 amino acids) made the product secreted into the medium. In conclusion, the hydrophobicity of 13 amino acids in length was enough for the GPI modification of the bovine liver 5'-nucleotidase.

Mutational analysis of the C-terminal signal peptide of bovine liver 5'-nucleotidase for GPI anchoring: a study on the significance of the hydrophilic spacer region.

Bovine liver 5'-nucleotidase is a GPI-anchored protein whose Ser523 attaches to GPI as the omega-site. For GPI-modification, pro-protein of the enzyme possesses a signal peptide at the C-terminus, comprising a hydrophilic spacer sequence of 8 amino acid residues and the following hydrophobic region of 17 amino acid residues. The C-terminal signal peptide is replaced by GPI on a luminal leaflet of endoplasmic reticulum. To characterize the C-terminal signal peptide for GPI modification, we constructed a series of deletion and elongation mutant genes, altering length of the hydrophilic spacer sequence by site-directed mutagenesis. Systematic deletion and Ala insertion of the sequence showed that the sequence of 6-14 residues were compatible for GPI modification. For GPI transfer to the pro-protein, the optimum length of spacer sequence would be 8, being consistent with natural selection. The spacer sequence may play a role for leading the omega-residue correctly to the active site of putative GPI transamidase. The elongation of the spacer is more permissible than deletion. Nevertheless, the length of the spacer sequence may influence efficiency of GPI modification by its positive or negative control.

Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties.

A sphingomyelinase was purified 980-fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)2SO4 precipitation and chromatography on CM-Sephadex, DEAE-cellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholipases. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (Mr = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.

Studies on lipase from Mucor javanicus. I. Purification and properties.

1. Lipase produced by a mold, Mucor javanicus, was purified about 180-fold from the ethanol precipitate of the culture filtrate. Purification was achieved by acid precipitation followed by gel filtrations on Sephadex G-200 (at low ionic strength) and Sephadex G-75 (at a high ionic strength). The purified enzyme preparation showed unusual behavior on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 21 000. The enzyme had a positional specificity towards the position 1 and 3 of triacylglycerols. 2. Lipase in the crude preparation takes an aggregated form. aggregated form was achieved by raising the ionic strength of the medium. 3. The purified lipase preparation from Mucor javanicus exhibits phospholipase A1 activity, hydrolyzing the carboxyl ester at the 1-position of phosphatidylcholine. This activity seems to be due to the action of the lipase itself and not due to any other specific phospholipases.

The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.

By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol caused a drastic decrease in enzymatic activity, whereas the sphingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity.

A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.

Characterization of aminopeptidase N from the brush border membrane of the larvae midgut of silkworm, Bombyx mori as a zinc enzyme.

Three GPI-anchored proteins, aminopeptidase N, alkaline phosphatase and alkaline phosphodiesterase I were released from the midgut brush border membrane of Bombyx mori by phosphatidylinositol-specific phopholipase C and the aminopeptidase N was purified to a homogeneous state. N-terminus and 6 internal sequences, one of which possessed part of zinc-binding motif, showed homology with those from other species. The zinc content in purified aminopeptidase N was estimated as approximately 0.72 mol/mol of the protein and 1,10-phenanthroline completely inhibited the enzyme activity, suggesting zinc requirement for the activity. The aminopeptidase N activity was inhibited not only by probestin and actinonin, but also strongly depressed by amastatin, while leuhistin and bestatin were less inhibitory. These suggest that the active site of aminopeptidase N might be structurally different from those of mammals. Calcium and magnesium ions stimulated the aminopeptidase N activity, but copper ion was rather inhibitory. Zinc ion showed bi-modal effect on the activity, i.e., stimulatory at low concentration, but inhibitory at higher than 100 microM. This inhibition was completely restored by EDTA. These results suggest that the aminopeptidase N possesses two zinc ion-binding sites with high and low affinity as essential and inhibitory one, as well as some regulatory metal-binding sites.

Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus.

Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes.

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I.

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut: a putative receptor for Bacillus thuringiensis CryIA toxin.

An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.

Determination of covalently bound myo-inositol in bovine erythrocyte acetylcholinesterase and porcine kidney alkaline phosphatase.

Bovine erythrocyte acetylcholinesterase and porcine kidney alkaline phosphatase were purified to a homogeneous state. By using gas chromatography-mass spectrometry, we demonstrated the presence of covalently bound myo-inositol in these purified enzymes. The quantitative data suggest that one molecule of myo-inositol is bound to each subunit of these enzyme proteins. The covalently bound inositol was removed from these enzyme molecules by deamination with nitrous acid, suggesting the possibility that myo-inositol is directly bound to amino sugar.

Analysis of PI (phosphatidylinositol)-anchoring antigens in a patient of paroxysmal nocturnal hemoglobinuria (PNH) reveals deficiency of 1F5 antigen (CD59), a new complement-regulatory factor.

FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C.

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.

Direct evidence of involvement of glycosylphosphatidylinositol-anchored proteins in the heavy metal-mediated signal delivery into T lymphocytes.

The biological significance of the action of glycosylphosphatidylinositol (GPI)-anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI-anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)-triggered signal delivery to T lymphocytes. Thiol-reactive HgCl2, a multi-potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy-1, a representative GPI-anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2 is diminished by the pre-treatment of cells with phosphatidylinositol-specific phospholipase C, which partially cleaved GPI-anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI-anchored proteins in the HgCl2-mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan-class A gene (PIG-A), and its restoration in a transfectant with PIG-A.

Delivery of an accessory signal for cell activation by exogenous phosphatidylinositol-specific phospholipase C.

Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI-specific phospholipase C (PIPLC) from Bacillus thuringiensis was shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex-induced signal delivery for cell activation. This was demonstrated for very early protein tyrosine phosphorylation, early c-fos transcription and late DNA synthesis. For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released from the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane-bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the T cell receptor function is discussed.