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Calcium-dependent binding of Escherichia coli alpha-hemolysin to erythrocytes

Thesis/Dissertation ·
OSTI ID:5736237
Alpha hemolysin (AH), a protein secreted by certain strains of Escherichia coli, causes lysis of erythrocytes (RBCs) and is cytotoxic for other cells. The primary structure of AH contains an eight amino acid sequence tandemly repeated 13 times near the C-terminus. These repeated sequences are essential for hemolytic activity. AH also requires an unknown modification by an accessory protein, Hly C, for hemolytic activity. The role of calcium in the interaction of Ah with RBCs was investigated using recombinant strains which produced active and inactive forms of the toxin. Hemolytic activity was calcium-dependent. Osmotic protection experiments and immunoblots of SDS-PAGE separated proteins from washed, toxin-treated RBCs showed that the binding of active AH to RBCs was calcium-dependent. Binding of active AH to RBCs increased the calcium permeability of RBC membranes and resulted in changes in membrane protein profiles. The changes in membrane proteins did not cause the lysis of the cells. These results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells. {sup 45}Ca-autoradiography of the recombinant hemolysins separated by SDS-PAGE and transferred to nitrocellulose showed that active AH bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences since a deletion hemolysin missing 11 of the 13 repeated sequences did not bind calcium. This deletion hemolysin was non-hemolytic and did not bind to RBC membranes. Hemolysin lacking the Hly C modification was also non-hemolytic and did not bind to RBC membranes. This unmodified AH contained the repeated sequences and bound calcium as efficiently as active AH.
Research Organization:
West Virginia Univ., Morgantown, WV (USA)
OSTI ID:
5736237
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METALS
AMINO ACID SEQUENCE
ANTIBODIES
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CALCIUM
CALCIUM 45
CALCIUM ISOTOPES
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ELEMENTS
ERYTHROCYTES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
HEMOLYSINS
INTERMEDIATE MASS NUCLEI
ISOTOPES
KINETICS
LYSIS
MATERIALS
MEMBRANES
METALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
PERMEABILITY
RADIOISOTOPES
REACTION KINETICS