Abstract
Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.
Highlights
Cytotoxic platelet cytosol by conventional and fast protein liquid effects of NO are thought to mbediated by intracellular iron chromatography techniques
Nitric oxide stimulates the auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a time and concentration-dependent manner with maximal effects after about 60 min
NAD+-freeenzyme is not inhibited by SIN-1, indicating the absolute requirementof NAD+ as the substrate of the ADP-ribosylation reaction
Summary
ADP-ribosyltransferases transfer only onesingle ADP-ribose Purification of the 39-kDa Protein-The 39-kDa protein was pugroup to the acceptor protein and are mosctlearly defined in rified using platelet-rich plasma from outdated blood samples prothe action of certain bacterial toxins on animal cells (5-8). After 100,000 X g centrifugation and ADP-ribosylation of the protein in order to follow purification (detection of radioactivity), the 50-90% (NH&O,) precipitation was collected, resuspended in 10. This article must be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate mM sodium phosphate buffer (pH 9.0), and desalted with a Sephadex G-25 column, at a flow rate of 2 ml/min. The protein was applied to a Q-Sepharose (fast flow) column and eluted with 10 mM sodium phosphate buffer, pH 9.0. Nitric Oxide and ADP-ribosylation exchange column S-Sepharose (fast flow) equilibrated with 10 mM sodium phosphate buffer (pH 7.0). Desalted using a Sephadex G-25 column and loaded to a blue Sepharose CL-4B column, equilibrated with 10mM sodium phosphate buffer. The trichloroacetic acid precipitate of the ADP-ribosylation reaction was washed with water-saturated ether, resuspended in 100 mM Hepes buffer pH
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