Hiroki Kondo

Synthesis and characterization of 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine as an artificial boundary lipid

The synthesis and characterization of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), are described. DDPC has two amide bonds instead of ester bonds of regular lecithins such as 1,2-dimyristoylphosphatidylcholine (DMPC). In differential scanning calorimetry (DSC) measurements, DDPC gave two endothermic peaks: one was at 18.0 degrees C (delta H = 10.74 kJ.mol-1) and the other at 23.0 degrees C (delta H = 12.91 kJ.mol-1). The former peak was sharp and considered to be the phase transition of the hydrocarbon region, while the latter was assigned to the melt of the hydrogen-belt formed by the amide groups of DDPC. Addition of DDPC to DMPC made the DMPC membrane less fluid in the region close to the surface, and significantly increased the reconstitution efficiency of glycophorin into the membrane. This effect of DDPC was much larger than that of naturally occurring lipid, sphingomyelin.

Two separate tyrosine protein kinases in human platelets

Tyrosine protein kinase activities were detected in the cytosolic fraction (PC‐TPK) and the particulate fraction (PM‐TPK) in human platelets using the synthetic peptide, E11G1, (Glu‐Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly) as a substrate. PC‐TPK and PM‐TPK were different in substrate specificities, divalent cation requirements and apparent M r values. These results strongly suggest that in platelets there exist at least two separate tyrosine protein kinases; one is present in cytosol and the other might be associated with membranes.

Liposomal membranes I. Chemical damage of liposomal membranes with functional detergent

The interaction and reaction between liposomal membrane and a functional detergent, N-hexadecyl-N-(imidazol-4-yl)methyl-N,N-dimethylammonium chloride hydroperchlorate (Im-I), have been investigated in conjunction with the leakage of bromothymol blue encapsulated as a marker in the bilayers of liposomes. Im-I carries an imidazole moiety and was expected to behave as a simple lipase model. The reaction with Im-I significantly enhanced the leakage of bromothymol blue encapsulated in the egg lecithin and dipalmitoyl phosphatidylcholine liposomes. During the course of reaction with Im-I, the formation of acyl-imidazole intermediate was clearly identified, which was certainly connected with the bromothymol blue release. From various kinetic results on bromothymol blue release and acyl-imidazole formation, it has been suggested that the bromothymol blue release from liposomal bilayer may be caused by the local and instantaneous decomposition of lipids when Im-I penetrates into the bilayer. However, it has also been demonstrated that the immediate reconstruction of liposomes retains the barrier function to protect against the further release of bromothymol blue.

Liposomal membranes. VII. Fusion and aggregation of egg lecithin liposomes as promoted by polysaccharides

Abstract Pullulan, one of polysaccharides, induces aggregation of egg lecithin multilamellar liposomes as detected by turbidity of the solution. The turbidity disappears upon treatment with pullulanase, indicating that the aggregation induced by pullulan is reversible. On the other hand, the turbidity induced on single-walled liposomes is not completely reversed by incubation with the enzyme. The phenomenon is interpreted as the involvement of irreversible fusion of liposomes. Thus, the enzyme digestion method provides a novel simple means to distinguish fusion of liposomes from aggregation.

High tyrosine protein kinase activities in soluble and particulate fractions in bone marrow cells

High tyrosine protein kinase activities were detected in soluble and particulate forms from bone marrow cells using synthetic peptide (Glu‐Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly) as a substrate. Total activity of tyrosine protein kinase was 2.4 times higher in the soluble fraction but the specific activity was slightly higher in the particulate one. Mg2+ or Mn2+ requirements of these two enzymes for maximal activity were quite different from each other. Physiological significance of these two forms of enzymes is briefly discussed.

An activated S6 kinase in regenerating rat liver

S6 kinase activity was increased in the regenerating liver 5 h after partial hepatectomy compared with sham-operated liver. The protein kinase activity was eluted from DE-52 column at approximately 250 mM NaCl and was not affected by known regulators of protein kinases. The S6 kinase was further purified by chromatography on peptide R1A13-Sepharose 4B and Sephadex G-150. The molecular weight of the enzyme was estimated to be 4.5 X 10(4) by gel filtration. The enzyme catalyzes the phosphorylation of whole histone, mainly H2B histone, at 75 mM Mg2+. These properties are similar to those of a proteolytically modified Ca2+/phospholipid-independent form of protein kinase C.

Liposomal membranes. xiv. fusion of liposomal membranes induced by polyisoprenoids as monitored by fluorescence quenching method

Fusion of the single-walled liposomes of egg phosphatidylcholine as induced by the polyisoprenoids such as solanesol, trans-ethyl decaprenoate (EDP), coenzyme Q10, and dolichol has been investigated adopting the fluorescence quenching method. Relative efficiency of the polyisoprenoids employed on the induced fusion of liposomes was a sequence of solanesol less than or equal to EDP much less than CoQ10, dolichol, which was consistent with the result previously obtained by the dye-release method.

Reversed micelles to mimic the active site of metalloenzymes

Abstract The structure and reactivity of cobalt(II), nickel(II), and copper(II) halides have been investigated in 0.20 M CTAX (X = Cl, Br) |CHCl3 reversed micelles. The former two metal ions adopt a tetrahedral configuration at low water concentrations in the micelle. The tetrahedral complexes are converted to octahedral aqua complexes by increasing the water concentration (solvochromism) or by lowering the temperature (thermochromism). Upon reaction with imidazole, the tetrahedral cobalt and nickel halide complexes also undergo a structural transformation into an octahedral configuration with imidazole coordination. At low water concentrations, copper halides form a polynuclear complex bridged by halide ions and these halogen bridges are easily broken upon addition of water or imidazole. The copper complexes produced by reaction with imidazole were deduced to be CuIm2X2 and CuIm4X2 at intermediate and high ligand concentrations, respectively. It was also found that the cupric ion in reversed micelles is readily reduced to the cuprous ion with 2-mercaptoethanol, and the cuprous ion is oxidized to the cupric ion by reaction with hydrogen peroxide.

Phosphorylation by protein kinase C of a synthetic heptapeptide bearing a lysine residue on the C terminal side of serine

A peptide, Ala-Ser-Gly-Ser-Phe-Lys-Leu, which corresponds to Ala103-Leu109 of Hl histone, was synthesized and tested as substrate for protein kinase C. The serine residue at position 4 was phosphorylated specifically. Another peptide lacking the lysine at position 6 was not phosphorylated by the same enzyme, indicating the importance of that basic residue as the recognition site for protein kinase C.

A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

Abstract The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O -sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.