In human erythrocyte membranes incubated with [adenylate‐32P]NAD the 36 kDa protein is predominantly labeled. The labeling is greatly stimulated by nitroprusside in the presence of dithiothreitol. We have purified the 36 k Da protein and identified this modification as crysteine‐specific mono(ADP‐ribosylation) because: (i) labeling occured only when [32P]NAD was replaced by adenine [U‐14C]NAD, but not by [carbonyl‐14C]NAD; (ii) treatment of the prelabeled protein with snake venom phosphodiesterase led to releasing 5′‐[32P]AMP; (iii) the bond between the protein and the nucleotide was hydrolyzed by HgCl2, but was resistant to hydroxylamine. The 36 kDa protein reacted on Western blots with two different monoclonal antibodies (MAbs) against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and was immunoprecipitated by both MAbs.

No abstract

Homogeneous preparations of D-glyceraldehydc.3-phosphate dehydrogenase purified from rabbit muscle were found to contain 0,2-0,7 moles of covalently bound phosphate per mole of the enzyme. With the use of anti-phospholyrosine antibodies, evidence was obtained that the enzyme is phosphorylated at tyrosine residues.o-Glyeeraldehyde-3-phosphate dehydrogenase; Endogenous phosphate; Phosphotyrosine; Antibody to phosphotyrosine

Nitric oxide was recently demonstrated to stimulate ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our studies on the effect of glyceraldehyde-3-phosphate (GA3P), the natural substrate of dehydrogenase activity of GAPDH. indicated GA3P to be another very potent activator of ADP-ribosylation of the enzyme. GA3P was able to activate ADP-nbosylation only m the presence of DTT. The action of GA3P was associated with inhibition of GAPDH dehydrogenase activity K, for GA3P was at least 50-fold lower and maximal activation was somewhat higher than these values for other aldehydes that were also able to enhance GAPDH ADP-ribosylation in the presence of DTT. ADP-ribosylation was blocked by carboxamidomethylation of the essential cysteme SH-group. The bond between the prelabeled protein and ADP-ribose was resistant to hydrolysis with hydroxylamine and HgCl?. suggesting that a lysme e-amino group is the target for ADP-ribosylation.