Production and characterization of murine monoclonal antibodies to polypeptide hormones and their fragments.

The diagnostic importance of monoclonal antibodies assays for peptide hormones in biological fluids and for histological peptide localization is rapidly increasing. In our laboratory a general protocol has been developed for immunization and fusion that has been proven very useful, with minor modifications, for generating monoclonal antibodies against insulin, calcitonin, adrenocorticotropin and parathyroid hormone. Our procedure offers the following advantages: 1) it requires a relatively low amount of antigen; 2) it takes only 16-20 days from the first immunization to the time of fusion; 3) it mostly generates IgG producing hybrids; 4) it involves few manipulations of the animals and no i.v. injections. The widely used methods utilized for peptide carrier conjugation, few guidelines for the choice of peptide fragments suitable as immunogens and some applications of antipeptide monoclonal antibodies will be briefly discussed.

Immunocytochemical characterization of mouse monoclonal ACTH antibodies with a note on staining conditions and control procedures.

Mouse monoclonal antibodies (MAbs) have been produced against porcine ACTH and tested for their immunocytochemical utility. Ten out of 12 MAbs reacted with formaldehyde-fixed human ACTH[1-39] and fragments thereof. Cytochemical fragment testing revealed that 6 of the 10 MAbs recognized epitopes in the vicinity of the region where porcine ACTH differs from mouse ACTH (amino acids 26, 29 and 31). Both tissue and cytochemical model data indicate that many of the MAbs detected porcine ACTH with somewhat higher potency than human and rat ACTH (rat ACTH[1-39] is identical to mouse ACTH[1-39]). MAbs Nos. 5, 8 and 12, in particular, revealed a highly satisfactory signal to noise ratio also in formalin-fixed, paraffin-embedded specimens. Most of the MAbs were potent in detecting both the high concentrations of ACTH congeners in corticotrophs and melanotrophs and the lower concentrations of such peptides in human antropyloric gastrin cells. Blocking of tissue endogenous peroxidase activity reduced reactivity towards the MAbs. This could be circumvented by use of biotinylated primary antibodies combined with avidin/streptavidin-alkaline phosphatase detection. Availability of MAbs and of the corresponding synthetic antigen also made some quantitative comparisons and analyses of appropriate control procedures possible.

Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry.

Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.

Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay.

A procedure is described for the production of calcitonin-specific hybridomas which involves primary and secondary immunization of Balb/c mice in the hind footpads with free, synthetic human calcitonin and cell fusion of lymphocytes from popliteal lymphonodes with a P3 x 63 myeloma line. This protocol offers the following advantages: (a) it is short and easy to perform, (b) it requires small amounts of unconjugated antigen, and (c) it gives a high yield of antigen-specific IgG-secreting hybridomas. Routine screening was carried out by ELISA on solid phase calcitonin and binding of the monoclonals to free antigen was studied with calcitonin linked to biotin through a 13 carbon atom spacer. Monoclonal couples capable of simultaneously binding calcitonin in solution were found by pairing in all possible combinations 25 purified antibodies, in their unlabelled and biotinylated form, in a checkerboard matrix experimental system. Of the over 30 positive pairs identified, four were used in a one-step enzyme immunoassay for calcitonin determination on microtiter plates in a concn range between 0.1 and 5 ng/ml. With the detecting monoclonal directly conjugated to peroxidase with a heterobifunctional crosslinker, the range of the assay with one monoclonal pair was between 25 and 1000 pg/ml with an 18 hr incubation at 4 degrees C.

A monoclonal-based, two-site enzyme immunoassay of human insulin.

A procedure is described for the efficient production of insulin-specific monoclonal antibodies, which involves primary and secondary immunization of BALB/c mice in the hind footpads with bovine or porcine insulin and fusion of lymphocytes from popliteal lymph nodes with a P3x63 murine myeloma line. With this protocol, over 200 positive hybrids were obtained from four separate fusions. Dissociation constants of 31 purified monoclonals, cross-reacting with human insulin, were determined by two different methods and ranged between 4 X 10(-10) and 2 X 10(-6) mol/l. 24 monoclonals were biotinylated, paired in all possible combinations and tested by ELISA for their capacity to simultaneously bind to human insulin in a two-site assay. More than 40 monoclonal pairs were found which formed a sandwich with the hormone. The development of a simple and rapid one-step enzyme immunoassay is described, which involves a first monoclonal bound to the wells of a microtiter plate and a second monoclonal conjugated to alkaline phosphatase. With this assay, insulin can be determined in a range between 0.08 and 7.5 ng/ml in 3-4 h.

The primary structure of subunit II of NADH dehydrogenase from bovine-heart mitochondria.

Subunit II (with a molecular mass of about 24000 dalton, approximately 24 kDA) of NADH dehydrogenase from beef heart mitochondria was [ 14C ]carboxymethylated and cleaved with CNBr and proteolytic enzymes. Sequence analyses of purified fragments suggest that the subunit is composed of a homogeneous polypeptide chain, containing just over 230 residues. The primary structure of this chain was established except for a 14-residue internal part which was only determined by composition. The amino acid sequence suggests that four cysteine residues are involved in the binding of an iron-sulfur cluster. The subunit contains no long hydrophobic segment, in contrast to structures often found in membrane proteins, but in agreement with a model where the functional unit of NADH dehydrogenase in the membrane is shielded by other intra-membrane proteins. The polypeptide has a weak similarity to the iron-sulfur binding region of ferredoxin and has interesting but possibly insignificant similarities to parts of previously compared flavin-linked enzymes.

Membrane topology of ATP synthase from bovine heart mitochondria and Escherichia coli.

The polypeptides exposed to lipids in the membranous F0 sector of the mitochondrial and Escherichia coli ATP synthases were labelled with radioactive photoreactive lipids. Highly resolving gel electrophoretic conditions were used in order to separate all the eighteen components forming the bovine heart mitochondrial enzyme. The hydrophobic labelling was performed on fully active and inhibitor-sensitive ATP synthases. In the mitochondrial enzyme prepared according to Serrano et al. (1976) [J. Biol. Chem. 251, 2453-2461] seven polypeptides of Mr 30500; 11500; 10500; 10000; 9500; 8500 and 4500 were labelled. The major amount of radioactivity was associated with the 30500-Mr component, which is thought to be the adenine nucleotide carrier. In the preparation of Galante et al., (1979) which almost completely lacks this component [J. Biol. Chem. 254, 12372-12378] nine polypeptides of Mr 25000; 21000; 11500; 10500; 10000; 9500; 9200; 8500 and 4500 were labelled. In the ATPase synthase from E. coli the major amount of labelling was associated with subunit b and only a minor portion with subunit c.

Interaction of rhodanese with mitochondrial NADH dehydrogenase.

NADH dehydrogenase is an iron-sulfur flavoprotein which is isolated and purified from Complex I (mitochondrial NADH: ubiquinone oxidoreductase) by resolution with NaClO4. The activity of the enzyme (followed as NADH: 2-methylnaphthoquinone oxidoreductase) increases linearly with protein concentration (in the range between 0.2 and 1.0 mg/ml) and decreases with aging upon incubation on ice. In the present work a good correlation was found between enzymic activity and labile sulfide content, at least within the limits of sensitivity of the assays employed. Rhodanese (thiosulfate: cyanide sulfurtransferase (EC 2.8.1.1) purified from bovine liver mitochondria was shown to restore, in the presence of thiosulfate, the activity of the partly inactivated NADH dehydrogenase. Concomitantly, sulfur was transferred from thiosulfate to the flavoprotein and incorporated as acid-labile sulfide. Rhodanese-mediated sulfide transfer was directly demonstrated when the reactivation of NADH dehydrogenase was performed in the presence of radioactive thiosulfate (labeled in the outer sulfur) and the 35S-loaded flavoprotein was re-isolated by gel filtration chromatography. The results indicated that the [35S]sulfide was inserted in NADH dehydrogenase and appeared to constitute the structural basis for the increase in enzymic activity.

Purification of three iron-sulfur proteins from the iron-protein fragment of mitochondrial NADH-ubiquinone oxidoreductase.

A fragment containing non-heme iron and acid-labile sulfide but little flavin can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents. This iron-protein fragment [Hatefi, Y., & Stempel, K. E. (1969) J. Biol. Chem. 244, 2350] has been resolved with detergents and ammonium sulfate fractionation into iron and acid-labile sulfide containing fractions, here called ISP-I and ISP-(II + III). ISP-I consists predominantly of a single polypeptide of molecular weight 75000. ISP-(II + III) consists predominantly of three polypeptides in equimolar concentrations with molecular weights of 49,000, 30000, and 13000. Treatment of the latter with sodium trichloroacetate followed by ammonium sulfate fraction results in separation of the 49000 molecular weight polypeptide from the two smaller subunits. Both of these subfractions (ISP-II and ISP-III, respectively) contain non-heme iron. The three iron-sulfur proteins have been characterized by their absorption spectra and iron and acid-labile sulfide contents. On the basis of the distribution of iron among the fractions obtained from chaotropic resolution of the NADH-ubiquinone oxidoreductase complex, a minimum of six or seven iron-sulfur centers are present in this enzyme.

Substrate binding affinity changes in mitochondrial energy-linked reactions.

The effects of uncouplers and valinomycin plus nigericin (in the presence of K+) were studied on the apparent Km for substrates and apparent Vmax of the following energy-linked reactions catalyzed by submitochondrial particles: oxidative phosphorylation, NTP-33Pi exchange, ATP-driven electron transfer from succinate to NAD, and respiration-driven transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate. In all cases, partially uncoupling (up to 90%) concentrations of uncouplers of valinomycin plus nigericin were found to decrease apparent Vmax and to increase apparent Km. Results plotted as ln (Vmax/Km) versus the concentration of uncouplers or ionophores showed a linear decrease of the former as a function of increasing perturbant concentration (i.e., decreasing free energy). Because Vmax/Km may be considered as a measure of the apparent first-order rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex, the results are consistent with the interpretation that membrane energization leads to a change in enzyme conformation with the resultant increase in enzyme-substrate affinity and facilitation of the reaction rate under consideration. The significance of these findings with respect to the mechanism of action of the energy-transducing systems studied is discussed.

Independent inhibitions of mitochondrial complex V by the adenosinetriphosphatase inhibitor protein and active-site modifiers.

The methyl 4-azidobenzimidate derivative of the naturally occurring ATPase inhibitor protein (IF1) of mitochondria binds to the beta subunits of soluble F1-ATPase upon photoactivation [Klein, G., Satre, M., Dianoux, A.-C., & Vignais, P. V. (1981) Biochemistry 20, 1339--1344]. A number of specific ATPase inhibitors, namely, 4-chloro-7-nitrobenzofurazan (NBF-Cl), efrapeptin, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), phenylglyoxal, aurovertin, tridentate ferrous bathophenanthroline, and octylguanidine (referred to hereafter as "artificial" inhibitors), are also considered to bind to the beta subunit, and there is strong evidence that the first three bind at the active site. Since the inhibition by IF1 of complex V ATPase activity can be reversed by incubation of the inhibited complex at pH 8.0, this system was used to investigate whether the inhibitions brought about by IF1 and the artificial inhibitors were independent, mutually interfering, or mutually exclusive. The experiments were carried out in two ways. (a) Complex V was first maximally inhibited by IF1. Then an artificial inhibitor was added and allowed to react. Excess artificial inhibitor was removed by precipitation of the doubly inhibited complex V with ammonium sulfate and resuspension in inhibitor-free buffer at pH 8.0. Incubation at pH 8.0 released the inhibition due to IF1. However, it was found that the factor that controlled reemergence of ATPase activity was the degree of inhibition exerted by the artificial inhibitor. When the artificial inhibitor was removed first (which was done by addition of dithiothreitol when the artificial inhibitor was NBF-Cl), then reemergence of activity depended on incubation at pH 8.0 to reverse the inhibition due to IF1. These results indicated that IF1-inhibited complex V could be independently inhibited by various artificial inhibitors. The artificial inhibitors used in this type of study were NBF-Cl, efrapeptin, aurovertin, FSBA, and phenylglyoxal. (b) Complex V was first treated with the artificial inhibitor (ferrous bathophenanthroline or octylguanidine) and then with IF1. Results showed that prior treatment of complex V with these inhibitors did not interfere with IF1 subsequently exerting maximal and reversible inhibition. The above results have been discussed in view of the recent finding that F1-ATPase contains two functional and interacting hydrolytic sites [Grubmeyer, C., & Penefsky, H.S. (1981) J. Biol. Chem. 256, 3718--3727].

Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins.

The low molecular weight NADH dehydrogenase which can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents consists of three subunits in equimolar ratio [Galante, Y. M., & Hatefi, Y. (1979) Arch. Biochem. Biophys. 192, 559]. The largest subunit (subunit I) can be completely separated from the other two (subunits II + III) by treatment with sodium trichloroacetate and ammonium sulfate fractionation. Both the subunit I and subunit II + III fractions contain iron and acid-labile sulfur. From visible and EPR spectroscopy and the iron and acid-labile sulfide content, we propose that the subunit II + III fraction contains a binuclear cluster. The cluster structure present in subunit I is as yet unclear. On separation of the subunits of NADH dehydrogenase, the FMN is lost.

Iron-sulfur N-1 clusters studied in NADH-ubiquinone oxidoreductase and in soluble NADH dehydrogenase.

Two N-1 type iron-sulfur clusters in NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) were potentiometrically resolved: one was titrated as a component with a midpoint oxidation-reduction potential of -335 mV at pH 8.0, and with an n-value equal to one; the other as an extremely low midpoint potential component (Em 8.0 less than -500 mV). These two clusters are tentatively assigned to N-1b and N-1a, respectively. Cluster N-1b is completely reducible with NADH and has a spin concentration of about 0.8/FMN. Its EPR spectrum can be simulated as a single rhombic component with principal g values of 2.019, 1.937, and 1.922, which correspond to the Center 1 reported earlier by Orme-Johnson, N. R., Hansen, R. E., and Beinert, H. (1974) J. Biol. Chem. 249, 1922-1927. At extremely low oxidation-reduction potentials (less than -450 mV), additional EPR signals emerge with apparent g values of gz = 2.03, gy = 1.95, and gx = 1.91, which we assign to cluster N-1a. It is difficult, however, to simulate the detailed spectral line shape of this component as a single rhombic component, suggesting some degree of protein modification or interaction with a neighboring oxidation-reduction component. EPR spectra of soluble NADH dehydrogenase, containing 5-6 g atoms of non-heme iron and 5-6 mol of acid-labile sulfide/mol of FMN, were examined. Signals from at least two iron-sulfur species could be distinguished in the NADH-reduced form: one of an N-1b type spectrum; the other of a spectrum with g values of 2.045, 1.95, and 1.87 (total of about 0.5 spin equivalents/FMN). This is the first example of an N-1 type signal detected in isolated soluble NADH dehydrogenase.

Mitochondrial adenosinetriphosphatase inhibitor protein: reversible interaction with complex V (ATP synthetase complex).

Mitochondrial ATPase inhibitor protein (IF1) reacts reversibly with complex V and inhibits up to 90% of its ATPase activity. Both the rate and extent of inhibition are pH and temperature dependent and increase as the pH is lowered from pH 8 tp 6.7 (the lowest pH examined) or as the temperature is increased from 4 to 36 degrees C. Nucleotide triphosphates plus Mg2+ ions are required for inhibition of complex V ATPase activity by IF1. In the presence of Mg2+ ions, the effectiveness order of nucleotides is ATP greater than ITP greater than GTP greater than UTP. Highly purified complex V, which requires added phospholipids for expressing ATPase and ATP-Pi exchange activities, cannot be inhibited by IF1 plust ATP-Mg2+ unless phospholipids are also added. This indicates that the active state of the enzyme is necessary for the IF1 effect to be manifested, because F1-ATPase, which does not contain nor require phospholipids for catalyzing ATP hydrolysis, can be inhibited by IF1 plus ATP-Mg2+ in the absence of added phospholipids. The IF1-inhibited complex V, but not IF1-inhibited F1-ATPase, can be reactivated by incubation at pH greater than 7.0 in the absence of ATP-Mg2+. The reactivation rate is pH dependent and is influenced by temperature and enzyme concentration. Complex V preparations contain small and variable amounts of IF1. This endogenous IF1 behaves the same as added IF1 with respect to conditions described above for inhibition and reactivation and can result in 25-50% inhibition in different complex V preparations. However, complex V lacking endogenous IF1 can be reconstituted from F0, F1, oligomycin sensitivity conferring protein, and phospholipids. Inhibition of this reconstituted preparation in the presence of ATP-Mg2+ depends entirely on addition of IF1. In general, the ATP-Pi exchange activity of complex V is more sensitive to the chemical inhibitors of F1-AtPase tha its ATPase activity. This is not so, however, for IF1. Under conditions that IF1 caused approximately 75% inhibition of ATPase activity of complex V, no more than 10% of the ATP-Pi exchange activity was inhibited.

Energy-linked transhydrogenation from NADPH to [14C]NADP.

Submitochondrial particles catalyze transhydrogenation from NADPH to [14C]NADP. This transhydrogenation is energy-linked, since its rate increases several-fold when the system is energized by succinate oxidation in the presence of rotenone (inhibitable by antimycin A or uncouplers), or by ATP hydrolysis (inhibitable by rutamycin or uncouplers). As in the case of transhydrogenation reactions from NAD(P)H to 3-ace-tylpyridine adenine dinucleotide phosphate and to thionicotinamide adenine dinucleotide phosphate, transhydrogenation from NADPH to [14C]NADP is also sensitive to treatment of the particles with trypsin or the arginyl residue modifier, butanedione. However, unlike the former reactions, transhydrogenation from NADPH to [14C]NADP cannot accumulate energy in the concentrations of the products, because, except for radioactivity, the nature and concentrations of the reactants and products remain unchanged throughout the course of the reaction. Therefore, the unrecoverable energy utilization by this region could be ascribed to an entropic component of the process, very likely an enzyme conformation change necessary for facilitation of hydride ion transfer from NADPH to [14C]NADP. This interpretation is in agreement with our previous kinetic evidence for enzyme conformation change associated with energy-linked transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate and thionicotinamide adenine dinucleotide phosphate, and with our conclusions regarding the mechanism of action of the transhydrogenase enzyme (Galante, Y.M., Lee, Y., and Hatefi, Y. (1980) J. Biol. Chem. 255, 9641-9646).

Energy-linked mitochondrial transhydrogenation from NADPH to NADP analogs.

The mitochondrial energy-linked transhydrogenase enzyme catalyzes hydride ion transfer between NAD and HADP, of which the reaction NADH leads to NADP is slow in the absence of energy and is accelerated 10-fold or more when the mitochondrial membrane is energized by ATP hydrolysis or respiration. The enzyme is a proton pump and effects proton translocation coupled to hydride ion transfer from NADPH to NAD (Earle, S.R., and Fisher, R.R. (1980) J. Biol Chem. 255, 827-830). The present studies have shown that submitochondrial particles also catalyze transhydrogenation from NADPH to two NADP analogs, namely 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP) and thionicotinamide adenine dinucleotide phosphate (thioNADP). Both reaction rates are greatly accelerated when the system is energized by ATP hydrolysis (inhibitable by uncouplers or rutamycin) or succinate oxidation (inhibitable by uncouplers or antimycin A). As in the case of NAD(H) in equilibrium with NADP(H) reactions, the transhydrogenations from NADPH to AcPyADP and thioNADP are inhibited by treatment of submitochondrial particles with trypsin or the arginyl residue modifier, butanedione. The Km values of the above substrates and the Vmax values under energy-linked conditions have been determined. The finding that the mitochondrial energy-linked transhydrogenase enzyme catalyzes transhydrogenation from NADPH to NADP analogs has revealed features regarding substrate site specificities and the effect of substrates on the directionality of proton translocation by the enzyme.