Tamara V. Bulargina

Nitroprusside stimulates the cysteine-specific mono(ADP-ribosylation) of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.

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.

Vasorelaxant and antiplatelet activity of 4,7-dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide: role of soluble guanylate cyclase, nitric oxide and thiols

Certain heterocyclic N‐oxides are vasodilators and inhibitors of platelet aggregation. The pharmacological activity of the furoxan derivative condensed with pyridazine di‐N‐oxide 4,7‐dimethyl‐1,2,5‐oxadiazolo[3,4‐d]pyridazine 1,5,6‐trioxide (FPTO) and the corresponding furazan (FPDO) was studied. FPTO reacted with thiols generating nitrite (NO), S‐nitrosoglutathione and hydroxylamine (nitroxyl) and converted oxyHb to metHb. FPDO did not generate detectable amounts of NO‐like species but reacted with thiols and oxyHb. FPTO and FPDO haem‐dependently stimulated the activity of soluble guanylate cyclase (sGC) and this stimulation was inhibited by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and by 0.1 mM dithiothreitol. FPTO relaxed noradrenaline‐precontracted aortic rings and its concentration‐response curve was biphasic (pIC50=9.03±0.13 and 5.85±0.06). FPDO was significantly less potent vasodilator (pIC50=5.19±0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb significantly inhibited only FPTO‐dependent relaxation. FPTO and FPDO were equipotent inhibitors of ADP‐induced platelet aggregation (IC50=0.63±0.15 and 0.49±0.05 μM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory effects of FPTO and FPDO were only partially blocked by sGC inhibitors. FPTO and FPDO (10–20 μM) significantly increased cyclic GMP levels in aortic rings and platelets and this increase was blocked by ODQ. Thus, FPTO can generate NO and, like FPDO, reacts with thiols and haem. The vasorelaxant activity of FPTO and FPDO is sGC‐dependent and a predominant role is played by NO at FPTO concentrations below 1 μM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.

Epitope mapping of anti‐troponin I monoclonal antibodies

Two groups of monoclonal antibodies (MAbs) specific to human cardiac troponin I (cTnI) were generated by immunization of mice by isolated cTnI (group I, 16 MAbs) or by the whole troponin complex (group II, 15 MAbs). Two sets of overlapping decapeptides covering the complete sequence of cTnI were prepared and used for epitope mapping by SPOT technique. Majority of MAbs (28 out of 31) interacts with synthetic peptides thus indicating that they recognize liner epitopes. MAbs raised against isolated cTnI preferentially recognize epitopes located at the N‐ or C‐terminal ends of cTnI. Nine out of fifteen MAbs raised against whole troponin complex interact with epitopes located in the N‐terminal part of cTnI. Generation of MAbs recognizing both isolated cTnI and cTnI inside of troponin complex and mapping their epitopes provides reliable detection of TnI in serum of patients with acute myocardial infarction.

D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE PURIFIED FROM RABBIT MUSCLE CONTAINS PHOSPHOTYROSINE

Homogeneous preparations of d‐glyceraldehyde‐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‐phosphotyrosine antibodies, evidence was obtained that the enzyme is phosphorylated at tyrosine residues.

Glyceraldehyde-3-phosphate activates auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase

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‐ribosylation only in the presence of DTT. The action of GA3P was associated with inhibition of GAPDH dehydrogenase activity. K a 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 cysteine SH‐group. The bond between the prelabeled protein and ADP‐ribose was resistant to hydrolysis with hydroxylamine and HgCl2, suggesting that a lysine ε‐amino group is the target for ADP‐ribosylation.

Interaction of a synthetic peptide of the interferon α-2 C-terminal part with human blood leukocytes: Binding to peripheral blood mononuclear cells

A biologically active synthetic peptide, 2438, representing the 124–138 amino acid sequence of the human interferon α‐2 (IFN α‐2) molecule, which is known to possess IFN‐like antiproliferative activity, specifically binds to human blood leukocytes. Scatchard plots reveal two different K fd values, for the ‘low’ and ‘high’ affinity binding. The interaction of the 125I‐labelled peptide 2438 with the cells is not impaired by human IFN α‐2 or cholera toxin.

Interaction of a synthetic peptide of the interferon α2 C-terminal part with human blood leukocytes. II. Effect on protein tyrosine phosphorylation

Tyrosine phosphorylation in human blood lymphocytes was studied as a function of stimulation with concanavalin A (ConA) and treatment of the cells with interferon α2 (IFNα2) and/or an IFN‐derived C‐terminal synthetic peptide 2438 (amino acid residues 124–138). Both IFNα2 and the peptide 2438 decreased the level of protein tyrosine phosphorylation in the ConA‐stimulated cells. In unstimulated cells, IFNα2 increased, and the peptide 2438 decreased the level of the tyrosine phosphorylation. A possible correlation of these effects with stimulation or cell proliferation is discussed.