Separation of solubilized A2 adenosine receptors of human platelets from non-receptor [3H]NECA binding sites by gel filtration.

Human platelet membranes were solubilized with the zwitterionic detergent CHAPS (3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and the solubilized extract subjected to gel filtration. Binding of the adenosine receptor agonist [3H]NECA (5'-N-ethylcarboxamidoadenosine) was measured to the eluted fractions. Two [3H]NECA binding peaks were eluted, the first of them with the void volume. This first peak represented between 10% and 25% of the [3H]NECA binding activity eluted from the column. It bound [3H]NECA in a reversible, saturable and GTP-dependent manner with an affinity of 46 nmol/l and a binding capacity of 510 fmol/mg protein. Various adenosine receptor ligands competed for the binding of [3H]NECA to the first peak with a pharmacological profile characteristic for the A2 adenosine receptor as determined from adenylate cyclase experiments. In contrast, most adenosine receptor ligands did not compete for [3H]NECA binding to the second, major peak. These results suggest that a solubilized A2 receptor-Gs protein complex of human platelets can be separated from other [3H]NECA binding sites by gel filtration. This allows reliable radioligand binding studies of the A2 adenosine receptor of human platelets.

8-Cyclopentyl-1,3-dipropylxanthine (DPCPX)--a selective high affinity antagonist radioligand for A1 adenosine receptors.

The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A1 adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A1 adenosine receptors and the stimulation via A2 adenosine receptors. The Ki-values of this antagonism were 0.45 nM at the A1 receptor of rat fat cells, and 330 nM at the A2 receptor of human platelets, giving a more than 700-fold A1-selectivity. A similar A1-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [3H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A1 receptors in membranes of bovine brain and heart, and rat brain and fat cells (KD-values 50-190 pM). Its nonspecific binding was about 1% of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [3H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A1 receptor.

Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen.

Sera from patients with primary biliary cirrhosis reacted with four major bands in beef heart mitochondria and ATPase extract when analyzed by immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These four immunologically reactive bands corresponded to protein bands with molecular weights of about (a) 80,000; (b) 63,000; (c) 56,000; and (d) 43,000 to 46,000. An additional immunoreactive band was found with some high-titered primary biliary cirrhosis sera at 36,000. No association with any ATPase subunits was found, except for band c which migrated between the alpha- and beta-subunit of ATPase. Most ATPase fractions did not contain this band c, indicating that M2 determinants, as defined by immunoblot, are not identical with any ATPase subunit. Species and nonspecies-specific determinants of M2 were identified using mitochondria from rat liver and human heart and liver. Antigenic bands a, c and d were nonspecies-specific. Band b and e occurred only in beef heart. An additional determinant at about 38,000 was detected using human heart and liver mitochondria. Primary biliary cirrhosis sera showed a typical reaction with two protein bands of Escherichia coli, one at about 85,000 to 90,000 and the other at 60,000. Antibodies against both determinants could be absorbed with submitochondrial particles of beef heart showing that E. coli shares cross-reacting determinants with mitochondria. Sera from 56 primary biliary cirrhosis patients were tested using beef heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of ATPase-associated antigen (M2) for detection of antimitochondrial antibodies in primary biliary cirrhosis by fluorometric immunoassay.

An indirect binding assay, the fluorometric immunoassay (FIAX), was established for the detection of anti-M2 antibodies which are specific markers for primary biliary cirrhosis (PBC). Submitochondrial particles (SMP) from beef heart and rat liver and the ATPase-associated antigen (M2) were used. The antigens were fixed to a cellulose acetate surface, SMP at a concentration of 2 mg/ml, ATPase at a concentration of 0.2 mg/ml. Sera were used at 1:60 and 1:120 and bound antimitochondrial antibodies (AMA) were demonstrated by fluorescent isothiocyanate labelled monospecific anti-human IgG, IgM and IgA antibodies. The fluorescent signals were proportional to the AMA titre in the serum samples and were measured in a fluorometer (FIAX 100). Of 94 patients with PBC, 92 had AMA against SMP from beef heart compared with 76 in the complement fixation test (CFT) and 84 in the immunofluorescence test (IFL). Ninety reacted with the ATPase-associated M2 antigen. Sera from patients known to have AMA of different specificities (anti-M1, anti-M3, anti-M5, anti-M6) reacted with SMP from beef heart and/or rat liver but not with M2.

ATPase-associated antigen (M2): marker antigen for serological diagnosis of primary biliary cirrhosis.

Serum samples from 94 patients with primary biliary cirrhosis (PBC) and 17 patients with chronic cholestatic hepatitis (CCH) were tested in the fluorometric immunoassay (FIAX) against the nonorgan-specific ATPase-associated antigen (M2) and against submitochondrial from beef heart and rat liver, to evaluate the specificity and sensitivity of the M2 antigen for the diagnosis of PBC. As controls serum samples from 42 patients with other antimitochondrial antibody (AMA) specificity (against M1, M3, M5, and M6) as well as samples from 417 patients with various other hepatic and non-hepatic disorders were used. Serum samples from 91 of the 94 PBC patients (97%) and all 17 with CCH reacted with the M2 antigen. However, when SMP from rat liver and beef heart were tested in parallel in the FIAX, AMA could be detected in all PBC serum samples. None of the 42 patients with different types of AMA had reactions with the M2 antigen but all had reactions with SMP from rat-liver or beef-heart mitochondria or both. Among the other 417 patients with hepatic and non-hepatic disorders only 4(1%), all with collagen diseases, had anti-M2 antibodies.

Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a component of the mitochondrial F1-ATPase complex.

The complement fixing antigen of the inner mitochondrial membrane previously shown to be associated with the mitochondrial ATPase could be further purified by subjecting the ATPase extracted from beef heart and brown fat mitochondria to ion exchange and gel filtration chromatography. Although the ATPase activity could be clearly dissociated from the complement fixing activity, subunits of the F1-ATPase complex were always found in the purified fractions. The alpha, gamma, delta and epsilon subunits of the complex could be excluded with high probability as target antigens in contrast to the beta band which was always found in association with the antigen activity. These findings imply that the active centre of the ATPase enzyme is not involved in the antibody reaction but molecules of the ATPase complex may have antigen binding capacity. Treatment of ATPase associated antigen with trypsin did not markedly affect the complement binding, while SMP's treated in the same way lost their antigen activity indicating that sera from patients with primary biliary cirrhosis (PBC) may have mitochondrial antibodies of different specificities reacting with trypsin sensitive as well as trypsin insensitive components of the inner membrane. The purified antigen reacted exclusively with sera from patients with PBC and may be therefore used as a marker antigen.