Measuring parathyroid hormone (PTH) in patients with oxidative stress--do we need a fourth generation parathyroid hormone assay?

Oxidation of PTH at methionine residues results in loss of biological activity. PTH may be oxidized in patients with renal disease. The aim of this study was to develop an assay considering oxidation of PTH. Oxidized hPTH was analyzed by high resolution nano-liquid chromatography coupled to ESI-FTT tandem mass spectrometry (nanoLC-ESI-FT-MS/MS) directly and after proteolytic cleavage. The oxidized hPTH(1-84) sample shows TIC-peaks at 18-20 min and several mass peaks due to mass shifts caused by oxidations. No significant signal for oxidized hPTH(1-84) species after removal of oxidized PTH molecules by a specific column with monoclonal antibodies (MAB) raised against the oxidized hPTH was detectable. By using this column in samples from 18 patients on dialysis we could demonstrate that measured PTH concentrations were substantially lower when considering oxidized forms of PTH. The relationship between PTH concentrations determined directly and those concentrations measured after removal of the oxidized PTH forms varies substantially. In some patients only 7% of traditionally measured PTH was free of oxidation, whereas in other patients 34% of the traditionally measured PTH was real intact PTH. In conclusion, a huge but not constant proportion of PTH molecules are oxidized in patients requiring dialysis. Since oxidized PTH is biologically inactive, the currently used methods to detect PTH in daily clinical practice may not adequately reflect PTH-related bone and cardiovascular abnormalities in patients on dialysis.

Plasma myostatin measured by a competitive ELISA using a highly specific antiserum.

BACKGROUND: Understanding the (patho)physiology of the negative muscle regulator myostatin (Myo) is important for patients with skeletal muscle disorders or cardiac disease. However, a reliable tool for measuring plasma Myo immunoreactivity is still lacking. METHODS: Human full-length proMyo was used to raise a polyclonal rabbit antiserum for a competitive Myo ELISA that was validated in patients with decompensated congestive heart failure (CHF) and in control patients (n=20 each). RESULTS: The Myo antiserum detected all subunits of human proMyo. The calibration curve showed an optimal range between 0.3 and 83.3 ng/ml (7.5-2100 pmol/l), with no cross-reactivity to growth differentiation factor-11, follistatin and follistatin-related gene protein. The inter-assay and intra-assay variances in human serum were ≤15% and ≤10%, respectively; the detection limit was 270 pg/ml (6.75 pmol/l). The assay showed excellent linearity in human plasma. Plasma NT-proBNP and Myo were significantly elevated in decompensated CHF compared with control patients and decreased significantly upon recompensating therapy. CONCLUSION: We describe the development of the first ELISA for myostatin immunoreactivity and its validation during recompensating therapy for CHF. This assay will be valuable for investigating neurological and cardiac diseases and states of cachexia, insulin resistance, and obesity.

Structure-activity relationship of an alpha-toxin Bs-Tx28 from scorpion (Buthus sindicus) venom suggests a new alpha-toxin subfamily.

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na(+), K(+), Ca(+) or Cl(-). An alpha-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3+/-2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain alpha-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJalpha-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na(+) channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known alpha- and alpha-like toxins suggests the presence of a new and separate subfamily of scorpion alpha-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD(50) 0.088 and 14.3microg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC(50)=0.01microg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na(+)-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical alpha-mammal (AaH-II), alpha-insect (Lqh-alphaIT), and alpha-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a "necklace-like" structure differing significantly in all alpha-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned alpha-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion alpha-mammal toxins.

Complete structure determination of N-acetyl-D-galactosamine-binding mistletoe lectin-3 from Viscum album L. album.

The primary structure of the B chain of the N-acetyl-D-galactosamine-recognizing mistletoe lectin-3 (ML-3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC-separation and Edman degradation and confirmation of the peptide sequences by MALDI-MS. ML-3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N-glycosylation sites at positions Asn(95) and Asn(135) of the lectin, were determined by a combination of glycosidase treatment and MALDI-MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type-II RIPs, although there are remarkable differences in the D-galactose-specific mistletoe lectin-1B chain. The recently published primary structure of the mistletoe lectin-3A chain1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML-3. The model demonstrates, unequivocally, that ML-3 is a member of the type-II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML-1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin-3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations.

Glycosylation of Rapana thomasiana hemocyanin. Comparison with other prosobranch (gastropod) hemocyanins.

The carbohydrate content and composition of hemocyanins (Hcs) of three prosobranchs (gastropods), Rapana thomasiana, Megathura crenulata and Haliotis tuberculata, were compared. The analyses were performed by gas-liquid chromatography after methanolysis, re-N-acetylation and trimethylsilylation. The two structural subunits of R. thomasiana Hc, RtH1 and RtH2, both showed 2.6% (w/w) carbohydrate content with very similar monosaccharide composition, indicative for N-glycosylation. The two isoforms of M. crenulata Hc (KLH), KLH1 and KLH2, on the other hand, definitely differed in glycosylation: KLH2 (3.4% carbohydrate, w/w) comprised relatively less mannose and more N-acetylgalactosamine than KLH1 (3.0% carbohydrate, w/w), in agreement with the fact that O-glycosylation has been observed in a functional unit (FU) of KLH2. For the Hc of the abalone H. tuberculata, with 4.5% (w/w) carbohydrate, appreciable amounts of 3-O-methyl-d-mannose and 3-O-methyl-d-galactose were detected, showing that the occurrence of methylated sugars is not restricted to the Hcs of pulmonates. From the structural subunit RtH2 of Rapana Hc the FUs RtH2-b and RtH2-d were isolated. On the basis of amino acid sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the respective native and PNGase-F-treated glycopeptides, one N-glycosylation site was found for each FU. This site was located at Asn-405 for RtH2-b and at Asn-394 for RtH2-d; the carbohydrate moiety corresponded to GlcNAc2Man6 and GlcNAc2Man5, respectively. A comparison was made with the N-glycosylation sites of other FUs of Rapana Hc.

Isolation and quantification of chitin-binding mistletoe lectin from mistletoe extracts and validation of this method.

A method was established to isolate and quantify small amounts of chitin-binding mistletoe lectin (cbML) from extracts of the mistletoe (Viscum album L.) by affinity and reverse phase high performance liquid chromatography. A validation, according to ICH guidelines, of this analytical method was carried out and showed that specificity, robustness and precision are guaranteed. In addition, linearity is ensured for a content between 0.6 and 4.1 microg/ml of cbML in the extracts and recovery was calculated to be in the range of 94 to 100%. So, accuracy of the method is guaranteed as well. As far as the range of the analytical method is concerned, a minimum of 1.2 microg and a maximum of 8.2 microg cbML can be incubated with the affinity material. Detection and quantitation limits were calculated to be 0.13 and 0.46 microg/ml cbML, respectively.

Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. album.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.

Adenosine binding sites at S-adenosylhomocysteine hydrolase are controlled by the NAD+/NADH ratio of the enzyme.

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine. On the basis of the kinetics of Ado binding to AdoHcy hydrolase we have shown that AdoHcy hydrolase binds Ado with different affinities [Kidney Blood Press. Res. 19 (1996) 100]. Since AdoHcy hydrolase in its totally reduced form binds Ado with high affinity we determined in the present study the Ado binding characteristics of purified AdoHcy hydrolase from bovine kidney (native form) and of reconstituted forms with defined NAD(+)/NADH ratios. AdoHcy hydrolase in its native form and at a ratio of 50% NAD(+) and 50% NADH exhibits two binding sites for Ado with a K(D1) of 9.2+/-0.6 nmol/L and a K(D2) of 1.4+/-0.1 micromol/L, respectively. Binding of Ado to AdoHcy hydrolase in its NADH form and in its NAD(+) form exhibits only one binding site with high affinity 48.3+/-2.7 nmol/L for the NADH form and with a low affinity of 4.9+/-0.3 micromol/L for the NAD(+) form. To identify these two Ado binding sites, AdoHcy hydrolase was covalently modified with [2-3H]-8-azido-Ado. After irradiation of the native AdoHcy hydrolase two different photolabeled peptides were isolated and identified as Asp(307)-Val(325) and Tyr(379)-Thr(410). When the reconstituted AdoHcy hydrolase in its NADH and in its NAD(+) form was irradiated with [2-3H]-8-azido-Ado only one peptide was identified as Asn(312)-Lys(318) from the NADH form and as Asp(391)-Ala(396) from the NAD(+) form. Based on the crystallographic data, the labeled peptide Asp(391)-Ala(396) (low affinity binding site), appears to belong to the catalytic domain of AdoHcy hydrolase, whereas the labeled peptide, identified as Asn(312)-Lys(318) (high affinity binding site), is located in the NAD domain. In conclusion, our data show that AdoHcy hydrolase has two different Ado binding sites which are dependent upon the enzyme-bound NAD(+)/NADH ratios.

The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity.

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.

A skeletal muscle troponin T specific ELISA based on the use of an antibody against the soluble troponin T (16-31) fragment.

Proteolytic degradation of muscle, which occurs post-mortem as part of the meat-ageing process, results in the production of protein fragments. In beef, degradation of skeletal muscle troponin T (TnT) results in the generation of a 16-residue long peptide (TnT (16-31)), identified in trichloroacetic acid (TCA) soluble extracts. We report the development of a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of TnT (16-31), using polyclonal antibodies raised against synthetic TnT (16-31). The ELISA procedure is based on inhibition of binding of the antibodies to immobilised TnT (16-31) by TnT (16-31) present in solution. Its useful range is 30 pmol to 2 nmol TnT (16-31)/ml. Quantification of TnT (16-31) in TCA muscle extracts showed that its concentration was enhanced with ageing. Moreover, a correlation between TnT (16-31) levels and meat tenderness was observed. The ELISA developed herein may prove advantageous for future use at the research and industrial level.

Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin.

The complete amino acid sequence of Rapana thomasiana hemocyanin functional unit RtH2-e was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin, and trypsin. The single-polypeptide chain of RtH2-e consists of 413 amino acid residues and contains two consensus sequences NXS/T (positions 11-19 and 127-129), potential sites for N-glycosylation. Monosaccharide analysis of RtH2-e revealed a carbohydrate content of about 1.1% and the presence of xylose, fucose, mannose, and N-acetylglucosamine, demonstrating that only N-linked carbohydrate chains of high-mannose type seem to be present. On basis of the monosaccharide composition and MALDI-MS analysis of native and PNGase-F-treated chymotryptic glycopeptide fragment of RtH2-e the oligosaccharide Man(5)GlcNAc(2), attached to Asn(127), is suggested. Multiple sequence alignments with other molluscan hemocyanin e functional units revealed an identity of 63% to the cephalopod Octopus dofleini and of 69% to the gastropod Haliotis tuberculata. The present results are discussed in view of the recently determined X-ray structure of the functional unit g of the O. dofleini hemocyanin.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect.

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.