Bovine superoxide dismutase: a radioimmunoassay.

Rabbit antibodies to bovine superoxide dismutase have been produced and used to develop a double-antibody solid phase radioimmunoassay for the enzyme. The assay is sensitive and highly specific for the bovine enzyme, showing no cross-reactivity with the murine or human superoxide dismutases. It has been applied to the quantitation of exogenous enzyme in serum and extracts of mouse cells and tissues.     

A copper chaperone for superoxide dismutase that confers three types of copper/zinc superoxide dismutase activity in Arabidopsis

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.     

Crystal structure of the copper chaperone for superoxide dismutase.

Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins. The 1.8 A resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains. The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake. The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis. In the crystal, two SOD1-like domains interact to form a dimer. The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone.     

A solid-phase radioimmunoassay for fibrinogen.

A solid-phase radioimmunoassay for fibrinogen has been developed utilizing [¹⁴C]-methylated fibrinogen as standard antigen and fibrinogen-specific antibodies covalently linked to Sepharose. Fibrinogen was [¹⁴C]-methylated by reductive alkylation using [¹⁴C] formaldehyde and sodium borohydride. The methylated fibrinogen was unaltered in clotting ability and antigenicity.The assay, an isotope dilution assay, is quantitative for picomole amounts of fibrinogen. It is specific for fibrinogen in homologous plasma and in the presence of a variety of other proteins.     

A direct radioimmunoassay for tonin.

A sensitive radioimmunoassay for the measurement of tonin is described. It is based on the use of antibodies produced in rabbits against highly purified tonin obtained from rat submaxillary gland. A radioiodinated enzyme with high specific activity was obtained by the chloramine-T method. Optimal conditions for radioimmunoprecipitation were established and the double-antibody method was used to separate bound and free tonin. The radioimmunoassay may be used to measure tonin in amounts as low as 150 pg. This radioimmunoassay was applied to the measurement of tonin in rat saliva and in homogenates of submaxillary glands. Excellent correlation was found between tonin activity measured fluorometrically and tonin concentration obtained by the radioimmunoassay.     

A radioimmunoassay for human plasma corticosterone.

A radioimmunoassay for human plasma corticosterone has been developed. Antiserum against corticosterone was produced in rabbits immunized with corticosterone-21-hemisuccinate conjugated to bovine serum albumin. The antiserum cross-reacted with progesterone, DOC and dehydrocorticosterone more than 20%. After the extraction with ether, and the separation by Sephadex LH-20 microcolumn chromatography, recovery was 51.2 +/- 12.1% in 50 assays. The mean coefficient of variation between assays was 7.7% and within assays was 8.6%. Human plasma corticosterone is measured readily by assaying aliquots of an ether extract of 0.05 to 0.1 ml of plasma after microcolumn chromatography. The mean plasma corticosterone concentration at 9 a.m. was 7.1 +/- 3.2 ng/ml in 45 normal subjects. Plasma corticosterone increased 5.2 times as much as basal values after ACTH injection, whereas radioimmunoassayed cortisol increased 2.4 times. On the other hand, plasma corticosterone decreased to 22.6% of basal values at four hours after 1 mg dexamethasone, whereas radioimmunoassayed cortisol decreased to 12.3% of basal values.     

Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.     

A radioimmunoassay for measuring alpha-amidating enzyme activity.

A sensitive alpha-amidating enzyme (alpha AE) assay using C-terminal glycine-extended substance P (SP-Gly) as a substrate was developed. The product, substance P (SP), was measured by a radioimmunoassay with specific polyclonal antibodies which recognize SP with an affinity 10,000-fold higher than that of SP-Gly. The sensitivity of the radioimmunoassay was 5 fmol. Enzyme activity could be readily detected with 25 ng alpha AE partially purified from the conditioned medium of rat medullary thyroid carcinoma CA-77 cells. The Km and Vmax values were 2.0 +/- 0.2 microM and 1.7 +/- 0.1 nmol/mg/min (mean +/- SE, n = 3), respectively. The assay enabled the kinetic characterization of alpha AE from a single rat pituitary homogenate. Optimal Cu2+ required was 30 microM and greater than 3 mM of ascorbate was needed for maximal enzyme activity. The sensitivity of this assay will aid efforts to examine the regulation of in vivo alpha AE activity.     

The amino-acid sequence of copper/zinc superoxide dismutase from swordfish liver. Comparison of copper/zinc superoxide dismutase sequences

The amino acid sequence of copper/zinc superoxide dismutase from swordfish (Xiphias gladius) liver has been determined by alignment of the tryptic peptides according to the known sequence of bovine erythrocyte copper/zinc superoxide dismutase. This alignment has resulted in the ligands to the copper (His-47, 49, 76 and 94) and the zinc (His-76, 85, 134 and Asp-97) being conserved in all the copper/zinc superoxide dismutases sequenced so far. Also conserved in the sequences are the cysteines forming the intrachain disulphide bridge (Cys-58 and 160) and the essential arginine (Arg-157). Comparison of the amino acid sequence of swordfish liver copper/zinc superoxide dismutase with the bovine, human, horse, yeast and Photobacterium leiognathi indicates that the swordfish enzyme has a high homology with the other eukaryotic enzymes. Low homology is, however, observed with the P. leiognathi enzyme.     

A characterization of copper/zinc superoxide dismutase mutants at position 124. Zinc-deficient proteins.

Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme. Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc. The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation. Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form. The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques. The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type. In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125. The behavior is similar to that of the other mutants in most respects. The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives. The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme. A full profile up to pH 10.5 was obtained for the mutants.     

A hybrid superoxide dismutase containing both functional iron and manganese

A hybrid superoxide dismutase containing functional Mn and Fe has been isolated from Escherichia coli. Streptomycin, which binds tightly to both the Mn- and the Fe-containing superoxide dismutases, had the expected effect on the electrophoretic and chromatographic behavior of the hybrid. Treatment of the hybrid with H2O2, which selectively inactivates the Fe-containing enzyme, resulted in partial inactivation accompanied by a resegregation of subunits, with the formation of active Mn-enzyme and inactive Fe-enzyme. A similar resegregation of subunits was observed when the hybrid was exposed to 2.5 M guanidinium chloride. Hybrids containing Mn or Fe could be generated in vitro by mixing the Mn-enzyme with the Fe-enzyme, removing metals with 8-hydroxyquinoline in the presence of 2.5 M guanidinium chloride, and then dialyzing against Mn(II) or Fe(II) salts. Ten per cent of the activity of the Fe-superoxide dismutases is resistant to H2O2, which correlates with its content of Mn. Since the activity remaining after exhaustive treatment with H2O2 exhibited the electrophoretic mobility of the Fe-enzyme, we concluded that some of the active sites of the Fe-enzyme were actually occupied by Mn. It should be noted, however, that for purposes of metal reconstitution experiments, a definite specificity was demonstrated. The Mn-enzyme was reconstituted with Mn(II), whereas the Fe-enzyme activity was recovered using only Fe(II). We propose that the Fe-superoxide dismutase may be heterogeneous and that 10% of its activity is actually due to a Mn-containing variant with the same electrophoretic mobility. Only the apohybrid enzyme regained enzymatic activity using both Mn(II) and Fe(II).     

A multinuclear copper(I) cluster forms the dimerization interface in copper-loaded human copper chaperone for superoxide dismutase

Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.     

A radioimmunoassay for chironomid hemoglobin

A double antibody, competitive radioimmunoassay was developed for the quantitation of stage-specific hemoglobin from the insect Chironomus thummi. The radioimmunoassay will detect as little as 150 picograms of Hb 3, a hemoglobin synthesized and secreted into the hemolymph of larvae during the 4th, but not the 3rd instar. The assay also detects cross-reacting hemoglobins purified from 4th instar larvae and in freshly laid eggs. Application of this radioimmunoassay is discussed in light of the cross-reacting Hbs in the insect.     

A coated-tube radioimmunoassay for beta-thromboglobulin

A solid phase radioimmunoassay for beta-thromboglobulin is presented which has the advantage of easy technical performance. The antibody was absorbed onto polystyrene tubes and the separation of free and bound radiolabelled tracer was performed by simply sucking the tubes empty after incubation. The assay is precise and accurate but not particularly rapid, since an overnight incubation is needed. Maximum assay sensitivity was found to be 5.6 ng/ml beta-thromboglobulin for plasma samples. Preliminary studies showed that the sensitivity can be increased by the use of a more dilute antiserum. The plasma concentration of beta-thromboglobulin in 37 normal subjects was 17 +/- 4 ng/ml. The analytical performance of the assay corresponds to a commercial liquid-phase kit used for reference.     

A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid