Ryo Taguchi

Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry.

A phospholipid mixture extracted from cultured cells was directly analyzed by capillary (Cap) liquid chromatography (LC)/electrospray ionization (ESI) mass spectrometry (MS). Using a quadrupole mass spectrometer, we analyzed positive molecular ions, negative molecular ions, positive fragment ions and negative fragment ions under four different functions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map, in which the first dimension is elution time and the second dimension is mass, proved useful. Consequently, four different maps can be obtained by each of four different functions. Among them, from negative fragment ions at high cone voltage in the negative ion mode, ions that originated from acyl fatty acid and phosphorylcholine, phosphorylethanolamine and cyclic inositol phosphate can be detected at specific elution times. The map from positive fragment ions at high cone voltage in the positive ion mode indicated ions such as diradylglycerol and derivatives of 1-alkyl or 1-alkenyl cyclic phosphatidic acid from phosphatidylethanolamine (PE), and phosphorylcholine from choline-containing phospholipids. The map produced from positive molecular ions indicated choline-containing phospholipids such as phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and PE. The map of negative molecular ions effectively indicated acidic phospholipids such as phosphatidylinositol. We were able to obtain more than 500 molecular species of phospholipids by this method within a few hours immediately after extraction from culture cells using a mixture of chloroform and methanol (2:1). In this context, we concluded that the combination of Cap-LC and ESIMS seems to be very effective in the analysis of phospholipid classes and their molecular species.

Phosphatidyl inositol-specific phospholipase C from Clostridium novyi type A

Abstract From the culture broth of Clostridium novyi type A, phosphatidyl inositol-specific phospholipase C was separated from the major part of phospholipase C (γ-toxin) which hydrolyzes phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin. Sodium deoxycholate stimulated the activity of phosphatidyl inositol phospholipase C. The concentration of sodium deoxycholate for maximal stimulation was 0.2% with 2 m m phosphatidyl inositol. Divalent cations (Mg2+, Ca2+, and Zn2+) were rather inhibitory above 10−3 m . Phosphatidyl inositol phospholipase C was not inhibited by EDTA or o-phenanthroline. When phosphatidyl inositol phospholipase C was incubated with rat liver slices, not only alkaline phosphatase but also 5′-nucleotidase was liberated into the soluble fraction.

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.

Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was identified in lipids bound to human serum albumin. A cPA fraction was extracted and purified from human serum albumin by use of a combination of preparative TLC and HPLC. Electrospray ionization mass spectrometry of the purified fraction showed molecular ions corresponding to cPA, which was composed of some different fatty acid species. The most abundant component was identified as palmitoyl-cPA by tandem mass spectrometry using collision-induced dissociation. These data have established that cPA is a naturally occurring lipid bound to human serum albumin.

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: Hydrolytic and hemolytic actions on erythrocyte membranes

Abstract Purified sphingomyelinase of Bacillus cereus was incubated with sheep erythrocytes at 37 °C, either in the absence or presence of divalent metal ions such as Mg 2+ and Ca 2+ . During incubation, the enzyme at first was adsorbed onto the surfaces of sheeps erythrocytes; then these cells became fragile to cold shock (hot-cold hemolysis). The enzyme further attacked sheep erythrocytes by hydrolyzing sphingomyelin, made these cells more temperature sensitive, and finally hemolyzed them during incubation at 37 °C. Mg 2+ stimulated both the hemolytic and hydrolytic activities of sphingomyelinase, while Ca 2+ was inhibitory. In the presence of both Mg 2+ and Ca 2+ , the processes of sphingomyelin hydrolysis and cell lysis were more accelerated than those in the presence of Mg 2+ alone. These divalent cations were essentially without effect on the adsorption of enzyme to erythrocytes. By scanning electron microscopy, it was revealed that sphingomyelinase of Bacillus cereus induced invagination of sheep erythrocytes in parallel with hydrolysis of sphingomyelin on the cell surfaces and finally transformed these cells into spherocytes.

Effects of metal ions on sphingomyelinase activity of Bacillus cereus.

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Kms for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.

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 acetylchol‐inesterase 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.

Complete deficiency of 20 KDa homologous restriction factor (HRF20) and restoration with purified HRF20

20 KDa homologous restriction factor (HRF20) is a membrane glycoprotein which inhibits formation of membrane attack complexes of homologous complement. Erythrocytes from a patient who is completely deficient in HRF20 were readily hemolyzed by homologous complement activated by sucrose or by acidification as in paroxysmal nocturnal hemoglobinuria (PNH). After incubating PNH erythrocytes (PNH-E) with purified HRF20, the cells were analyzed by flow cytometry using a monoclonal antibody to HRF20 and shown to have the antigen absorbed. These PNH-E acquired resistance to hemolysis by homologous complement suggesting that HRF20 may be successfully used for treatment of these patients.