1990 Fiscal Year Final Research Report Summary
Molecular Structures of Bacterial Phospholipases C and Their Actions Toward Biomembranes
Project/Area Number |
63480465
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Research Category |
Grant-in-Aid for General Scientific Research (B)
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Allocation Type | Single-year Grants |
Research Field |
Biological pharmacy
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Research Institution | Nagoya City University (1988, 1990) Kobe University (1989) |
Principal Investigator |
IKEZAWA Hiroh Nagoya City Univ., Fac. Pharm. Sci., Professor, 薬学部, 教授 (40080163)
|
Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Tomoko Nagoya City Univ., Fac. Pharm. Sci., Assistant, 薬学部, 助手 (70214533)
TOMITA Masahiro Nagoya City Univ., Fac. Pharm. Sci., Lecturer, 薬学部, 講師 (20183494)
TAGUCHI Ryo Nagoya City Univ., Fac. Pharm. Sci., Assistant Professor, 薬学部, 助教授 (20080210)
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Project Period (FY) |
1988 – 1990
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Keywords | Bacterial Phospholipases C / Molicular Structures Of Phospholipases C / The Action Of Phospholipase C Toward Biomembranes / PI-anchored Membrane Proteins / Erythrocyte Membrane |
Research Abstract |
Using CD spectrum and prediction by computer analysis, it became apparent that the secondary structure of Bacillus cereus sphingomyelinase (sphingomyelin-hydrolyzing phospholipase C) contained essentially none of alpha-helix. Also, the content of beta-structure must be very low. One-hundred and fifty amino acid residues, amounting to approximately half of the enzyme protein, might contribute to the formation of loop and turn structures. Hydropathy analysis revealed the presence of hydrophobic domain consisting of 19 amino acid residues proximate to the N-terminus. This hydrophobic domain must be responsible for the affinity of sphingomyelinase to biomembranes. By chemical modification, carboxyl groups of Asp and Glu residues proved to exist in the active site. Also, there is a cystine bridge whose cleavage by DTT results in inactivation of sphingomyelinase. As regards phosphatidylinositol-specific phospholipase C, the cloning of cDNA is in progress, using peptide probes. The difference between Mg^<2+> and Mn^<2+> was examined in the activating effect on the action of Bacillus cereus sphingomyelinase toward bovine erythrocyte membrane. Also, 22-R-hydroxycholesterol proved to enhance the hemolytic action of sphingomyelenase toward bovine erythrocytes as well as the hydrolysis of membranous sphingomyelin by the enzyme, while melittin promoted only the hot hemolysis. Among eucaryotic PI-anchored proteins, we have determined the primary structure of 5'-nucleotidase from bovine liver and confirmed the sequence of a C-terminal hydrophobic peptide consisting of 25 amino acid residues to be the signal for attachment of the PI-anchor precursor to the protein. In the translocation from the plasma membrane to lysosomes. 70% of chicken liver 5'-nucleotidase was transformed into deanchored soluble form (s). We have a also purified and characterized aminopeptidase, a PI-anchored enzyme from midgut of silkworm. Bombyx mori.
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Research Products
(7 results)