Reduction of background radiation effects for positron lifetime measurements in the slow positron beamline at the Kyoto University Research Reactor.

Positron annihilation lifetime spectroscopy (PALS), which is recognized as one of the major analytical methods of positron annihilation spectroscopy, can directly detect information related to the size of vacancy-type defects from lifetime values. PALS measurements performed under high background radiation have been previously reported. It is well known that coincidence techniques such as age-momentum correlation (AMOC) measurements are effective for the background reduction, but count rates decline significantly. In this study, a preliminary experiment was performed to reduce the influence of the background radiation without the coincidence technique in the pulsing system of the Kyoto University research Reactor (KUR) slow positron beamline. This experiment involved the introduction of a gate circuit for the background radiation discrimination using a dynode signal from a single scintillation detector (photomultiplier). After introducing the gate circuit, the time resolution and the lifetime value of Kapton were 308 ps and 388 ± 3 ps, respectively, with count rates of ∼400 counts/s at a KUR 5 MW operation. In the AMOC measurement, the time resolution and the lifetime value of Kapton were 297 ps and 380 ± 7 ps, respectively, with count rates of ∼40 counts/s at a KUR 5 MW operation. When the single detector with the gate circuit was used, the count rate was ∼1 order of magnitude higher than those of the AMOC measurements, while the time resolutions of the two methods were comparable.

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.

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.

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.

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.

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.

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.

Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1.

Fission yeast lsd1 strains show aberrant mitosis with a lsd phenotype, large and small daughter nuclei, and a very thick septum, the phenotypic expression being temperature-sensitive. The lsd1(+) gene is the homologue of the budding yeast FAS2 gene encoding the fatty acid synthase alpha-subunit as reported previously (S. Saitoh, K. Takahashi, K. Nabeshima, Y. Yamashita, Y. Nakaseko, A. Hirata, M. Yanagida, J. Cell Biol. 134 (1996) 949--961). In this paper, lsd1 is considered to represent fas2. Here, three fas2 strains were investigated and found to have missense point mutations at different sites in the gene encoding the alpha-subunit of fatty acid synthase. The mutation affected only slightly the enzymatic activities monitored in vitro. Unexpectedly, abnormal phospholipids, phosphatidylcholine and phosphatidylethanolamine, both of which contain a very-long-chain fatty acyl residue (1-melissoyl-2-oleolyl-sn-glycero-3-phosphocholine and 1-melissoyl-2-oleolyl-sn-glycero-3-phosphoethanolamine), accumulated in fas2 strains in a temperature-sensitive manner. Rescue of the fas2 strains by addition of palmitate to the medium at restrictive temperature was accompanied by disappearance of these abnormal phospholipids. Accumulation of these lipids in membranes may cause alteration of various cellular functions.

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.

Pericardial fat accumulation in men as a risk factor for coronary artery disease.

An increment of abdominal visceral fat accumulation has been reported to be a coronary risk factor. We determined the predictive power of pericardial fat (Pfat) accumulation as intra-thoracic visceral fat, in the diagnosis of coronary artery disease (CAD). Among 251 (181 non-obese [body mass index<25], 70 obese [body mass index> or =25]) Japanese male patients who underwent computed tomography (CT), 128 (90 non-obese, 38 obese) patients were suffering from CAD. Pfat volume was determined by the sum of cross-sectional images 1cm thick from the atrial appendage to the apex over the diaphragm. Abdominal visceral fat (Vfat) and subcutaneous fat (Sfat) areas were measured by a single scan at the L4-L5 region. Pfat was most associated with Vfat in body fat distribution. In non-obese patients, Pfat was most associated with CAD among the various risk factors including body fat distribution. Moreover, Pfat was the strongest independent variable for the severity of CAD, determined by coronary angiogram. This result showed that pericardial fat accumulation was a stronger coronary risk factor than the other body fat distributions in non-obese men.

Effects of phospholipases C on the beta-receptor-adenylate cyclase system of chick erythrocyte membranes.

The beta-adrenergic receptor located in chick erythrocyte membranes was characterized using (-)-[3H]-dihydroalprenolol ([3H]-DHA) with rapid filtration techniques. The affinity of beta-adrenergic antagonist, (-)-propranolol, was approximately 100-fold higher than that of (+)-propranolol. Catecholamines were bound with the receptor in the following order, (-)-isoproterenol greater than (-)-norepinephrine greater than (-)-epinephrine, suggested the binding site to be beta 1-classification. When the membrane preparation was treated with phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens or phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis, [3H]-DHA binding was decreased to the level of 66 or 86% of the control, respectively. The treatment with sphingomyelinase C (SMase) of Bacillus cereus, however, did not cause any appreciable reduction of [3H]-DHA binding. Throughout these experiments, the equilibrium dissociation constant (KD) of [3H]-DHA was not influenced by phospholipases C. The affinity of isoproterenol for beta-receptor was decreased in the absence of GTP, but not altered in the presence of GTP by PIase action. Treatment with PCase or SMase, however, did not affect the affinity of isoproterenol for beta-receptor. Treatment with PIase inhibited basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activities. On the other hand, PCase treatment inhibited only isoproterenol-stimulated adenylate cyclase activity, but not basal and forskolin-stimulated activities. These results suggest that membrane phospholipids, especially phosphatidylcholine (PC) and phosphatidylinositol (PI), are directly related to the receptor binding and that PI interacts with adenylate cyclase activity.

T cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1.

We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis. Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations. In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes. When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC. We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids. The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor. In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase C from Clostridium novyi type A. II. Factors influencing the enzyme activity.

The apparent activity of phospholipase C[EC 3.1.4.3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates. When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5 mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5 mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation. The isoelectric point of the enzyme was 7.1 +/- 0.1.

Hydrolytic action of phospholipases on bacterial membranes.

Substrate specificities of phospholipases C[EC 3.1.4.3] from Clostridium novyi, Clostridium perfringens, Bacillus cereus, and Pseudomonas aureofaciens were studied under the same conditions. Phospholipases C from Clostridium novyi and Bacillus cereus show wide substrate specificities while those of Clostridium perfringens and Pseudomonas aureofaciens show relatively narrow specificities. On the basis of these results, the hydrolytic actions of these phospholipases on membrane lipids of Escherichia coli, Bacillus cereus, and Clostridium novyi were examined under the same conditions. The enzymes of Clostridium novyi and Bacillus cereus attacked all the membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine, phosphatidylglycerol, lyso-phosphatidylethanolamine, and o-aminoacylphosphatidylglycerol. Phospholipase C from Pseudomonas aureofaciens attacked these three membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine. Phospholipase C from Clostridium perfringens hardly attacked the phospholipids of these bacterial membranes. However, phospholipase C from Clostridium perfringens hydrolyzed phosphatidylethanolamine in a mixture containing lipid extract from Escherichia coli membrane and purified phosphatidylcholine from egg yolk.