We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.
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.
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 cTnl 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 acutc myocardial infarction. BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONALstructure of skeletal and cardiac TnI [4,5]. This makes possible production of cTnl specific antibodies which can be used for detection of many types of myocardial cells damage [6,7]. After cell death caused by infarction cTnI is released into the extracellular space and 4 -6 hours after onset of the chest pain can be detected in the blood stream [8,
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
1 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. 2 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. 3 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. 4 FPTO relaxed noradrenaline-precontracted aortic rings and its concentration-response curve was biphasic (pIC 50 =9.03+0.13 and 5.85+0.06). FPDO was signi®cantly less potent vasodilator (pIC 50 =5.19+0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb signi®cantly inhibited only FPTO-dependent relaxation. 5 FPTO and FPDO were equipotent inhibitors of ADP-induced platelet aggregation (IC 50 =0.63+0.15 and 0.49+0.05 mM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory eects of FPTO and FPDO were only partially blocked by sGC inhibitors. 6 FPTO and FPDO (10 ± 20 mM) signi®cantly increased cyclic GMP levels in aortic rings and platelets and this increase was blocked by ODQ. 7 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 mM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.
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