CNBr Fragments Research Articles

Biochemical Comparison of Rabbit Skeletal Muscle Alpha and Beta Tropomyosin Isoforms

The alpha and beta isoforms of tropomyosin (39 amino acid substitutions) were isolated from adult rabbit skeletal muscle without chemical modification. The levels of phosphorylation as judged by 2D PAGE were ∼10% (alpha) and nil (beta). Solutions of homodimeric beta tropomyosin (pH 7; ionic strength, ∼30 mM) display greater viscosity than those of alpha. Conversely, the enhancement of viscosity of the alpha isoform by skeletal troponin is greater than for beta. Mixtures of tropomyosin and the amino-terminal CNBr fragment of troponin-T, CB1 (res 1 - 151), were chromatographed on a size-exclusion column in the presence of different concentrations of KCl. In 0.1M salt, CB1 complexes and co-elutes with each isoform but is dissociated from either in 0.22 M. At intermediate salt, different degrees of complexation are observed - more extensive with alpha than beta. Further, the alpha-isoform elutes later in the salt gradient than the beta isoform during chromatography on skeletal troponin-Sepharose 4B. Tropomyosin isotype also has a bearing on thin filament regulation of the steady-state rate of actomyosin-S1 MgATPase. Reconstituted skeletal thin filaments composed solely of beta produce ∼30% less Ca(II)-induced activation compared to only alpha. This is observed at a high S1/actin ratio (6 uM myosin-S1A1, 3 uM thin filaments, pCa 4) and as a function of pCa (0.3 uM myosin-S1A1, 25 uM thin filaments).

Purification and functional characterization of complement C3 and a novel zymosan-binding protein in tilapia serum

Zymosan, a yeast cell wall preparation that binds activated forms of complement C3, is a useful model target to activate the complement system. In our trial to analyze C3 diversity in Nile tilapia at the protein level using zymosan, we found that a novel 240-kDa serum protein (ZBP-240) also bound to zymosan in addition to C3-derived fragments. In the present study, we aimed to characterize tilapia C3 and ZBP-240, focusing on their immune-related functions. Four distinct C3 isoforms were purified from tilapia serum and shown to possess an intrachain thioester bond. ZBP-240 was also isolated from tilapia serum and examined for its binding properties to various microbial targets. As a result, ZBP-240 showed a wide spectrum of binding to Gram-positive and Gram-negative bacteria and yeasts. Amino acid sequence analysis of CNBr fragments of ZBP-240 suggested that this is a novel protein with no homologous sequence in protein databases. It was also suggested that the binding of ZBP-240 to microbes largely depends on hydrophobic interactions in a divalent-cation-independent manner, and that there may be a divalent-cation-dependent factor that enhances the binding of ZBP-240 in tilapia serum. Interestingly, ZBP-240 showed opsonic activity for tilapia kidney phagocytes at a level comparable to that of C3, implying that ZBP-240 is a novel teleost opsonic serum protein.

Characterization of the Ubiquinone Binding Site in the Alternative NADH-Quinone Oxidoreductase of Saccharomyces cerevisiae by Photoaffinity Labeling

The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we conducted photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of the least possible modification of the substituents on the quinone ring. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ ring of 2 to be specifically cross-linked to the Phe281-Met410 region (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys-C) gave approximately 8 and approximately 4 kDa peptides, respectively. The approximately 8 kDa V8 digest was identified as the Thr329-Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the approximately 4 kDa Lys-C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the Gly374-Lys405 region (32 amino acids). Taken together, the binding site of the Q ring of 2 must be located in a common region of the V8 protease, and Lys-C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a three-dimensional structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD.

The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement

Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intramolecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p > 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.

The Complete Amino Acid Sequence of Green Turtle (Chelonia mydas) Egg White Ribonuclease

Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64-65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.

Primary and Tertiary Structure Studies of p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.

Exploring the Role of a Unique Carboxyl Residue in EmrE by Mass Spectrometry

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.

Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk: INVOLVEMENT OF RESIDUES 66–76 OF STRAND D IN THERMAL DENATURED β-LACTOGLOBULIN

beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degrees C. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degrees C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

T cell recognition pattern of bovine milk α S1-casein and its peptides

T cell recognition patterns of CAS1_Bovin, its limited hydrolysis, oxidized, reduced/alkylated, cyanogen bromide cleavage fractions and synthetic peptides were examined. Thirteen overlapping peptides covering the intact molecule, with chain lengths varied between 17 and 20 AA, were prepared by f-moc SPPS. In addition, six CNBr-cleavage fragments were obtained and extensively purified using RP/HPLC. Likewise, chemically modified derivatives and limited pepsin hydrolysate, were performed and the specificities were confirmed. Stimulation of PBMC and TCL cultures by the intact CAS1_Bovin molecule, synthetic peptides and modified derivatives were screened by [methyl- 3H] thymidine incorporations. PBMC phenotype was performed by flow cytometry and the mean CD4 +/CD8 + ratio of freshly prepared PBMC was compared with the ratio following specific CAS1_Bovin stimulation. CD4 + phenotypes (T H1/, T H2 and T H0) were assigned by assay of four marker cytokines IL-4, IL-6, IL-10 and IFN-γ. Five CNBr fragments and seven of the thirteen tested peptides were recognized by specific TCL. The most reactive epitopes of CAS1_Bovin comprised seven motifs namely: peptides Cas 1–18, Cas 16–35, Cas 67–85, Cas 91–110, Cas 136–155, Cas 152–169 and Cas 166–183. The stimulation range for the seven peptides was 1058–2383 cpm. Stimulation for the CNBr fragments were, respectively, 8670, 5808, 3324, 5465, 2255 and 321 cpm. Cytokine assay showed that CD4 + T H2 phenotype was dominant for half the number of patients, while T H1 solely or combined T H0 were represented in the other four cell culture filtrates. The T cell reactive epitopes described and their antibodies will be useful tools for methods in progress for the detection of masked casein epitopes encompassed in processed food. In conclusion, T cell recognition pattern of CAS1_Bovin was examined using extensively purified synthetic peptides and CNBr fragments. Five large and seven small peptides were clearly recognized. Peptides of chain length less than six AA were left unrecognised. CD4 + T H2 phenotype was the most dominant TCL subpopulations found in atopic patients while CD4 + T H1 was representative in the non-IgE mediated type IV hypersensitivity.

Multiple forms of medicinal leech destabilase-lysozyme

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2 Da, each of which contains seven disulfide bridges. Only one mass (12839.7 Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.

Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70 kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolytic as well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the Aα-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala 14 and Leu 15. Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.

Ferredoxin:NADP+ Oxidoreductase Is a Subunit of the Chloroplast Cytochrome b6fComplex

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.

Fibrin-mediated plasminogen activation.

Fibrin, but not fibrinogen, enhances the rate of activation of plasminogen by tissue type plasminogen activator (t-PA). Studies with enzymatic and chemical fragments of fibrinogen showed that several sites in fibrinogen are involved in this rate enhancement; these are, A alpha 148-160 (located in CNBr fragment FCB-2), and FCB-5 (a CNBr fragment comprising gamma 312-324), and recently discovered sites in the fibrinogen alpha C domains. All these sites are buried in fibrinogen, but exposed in fibrin and some fibrinogen fragments. For the first two of these, located in the D-domains, this was shown by the fact that monoclonal antibodies against A alpha 148-160 and gamma 312-324 bind to fibrin and rate enhancing fibrin(ogen) fragments, but not to fibrinogen. Direct binding studies indicate that at physiological concentrations plasminogen binds to FCB-2, and t-PA to FCB-5. More detailed studies have demonstrated the importance of residues A alpha-157 and A alpha-152, and that the minimum stretch with rate enhancing properties is A alpha 154-159. The sites in the alpha C domains await further identification. With the recently reported three-dimensional structure of fragments D and D-dimer it is now possible to explain these findings at the molecular level. Molecular calculations and experimental data show that the site A alpha 148-160 in fibrinogen is covered among others by a part of the A alpha chain (A alpha 166-195) that forms an alpha-helix, and by a globular domain formed by the beta-chain. On fibrin formation, the last two may move away, and give access to A alpha 148-160. It is conceivable that in the alpha C domain sites are involved in the early phases of fibrinolysis. The site A alpha 148-160 and that in FCB-5 may be more important at later stages. It is also clear that fibrin polymerization is important. This polymerization has probably several effects: exposure of the rate enhancing sites; mutual positioning of the t-PA and plasminogen binding sites; a concentrating effect of t-PA and plasminogen on the fibrin surface; effects on the kinetic properties of t-PA and plasminogen. These effects together explain the rate enhancement.

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.

Improved Accuracy in the Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Determination of the Molecular Mass of Cyanogen Bromide Fragments of Proteins by Post-Cleavage Reaction with Tris(Hydroxymethyl)Aminomethane

In order to improve accuracy in the matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) determination of the molecular mass of CNBr fragments of proteins, the post-cleavage reaction of these fragments with tris(hydroxymethyl)amino methane was tested. Mixtures of homoserine and homoserine lactone peptide fragments originating from CNBr cleavage of cytochrome c, lysozyme and human serum albumin were used as model compounds. Reaction of these fragments with tris(hydroxymethyl) aminomethane converts quantitatively the homoserine lactone ending peptides into the corresponding amides, leaving unmodified the homoserine ending forms. Thus, pairs of fragments which differ by 103 Daare formed. In contrast to the unmodified CNBr mixtures of peptides, which, due to the overlap of the signals of the free homoserine and homoserine lactone forms, produce unresolved peaks in the high mass region of the MALDI spectra, these pairs of fragments give resolved peaks up to a mass doublet of 20,000 Da. This permits accurate determination of the molecular mass of the fragments. Using this procedure, differences lower than 5 Da with respect to the calculated values were obtained for the fragments examined.

Biochemical and immunological properties of human cardiac troponin I fragments.

Cardiac troponin I (cTnI) is the inhibitory subunit of the troponin complex and is a biochemical marker for myocardial infarction (MI). It is found in human serum within 4-6 h following MI. One of us has shown [Morjana (1998) Biotechnol. Appl. Biochem. 28, 105-111] that MI patient serum TnI is cleaved at the N- and C-terminals and that the TnI fragments exist as a complex with tropinin C (TnC) and troponin T (TnT). In the present study, we have generated C-terminal truncated TnI fragments and studied their immunological and biochemical properties. Human recombinant TnI (rTnI) expressed in Escherichia coli is cleaved into a major fragment with a molecular mass of 17500 Da using CNBr. The major CNBr fragment contains the first 153 amino acids of human cTnI (TnI153). Cleavage of the rTnI with the endoproteinase Asp-N generates a smaller TnI fragment (TnI88, residues 6-96). TnI153 has higher immunological activity than that of rTnI and lower activity than that of TnI88, as judged by the Stratus II TnI Immunoassay. TnI153 exhibits biochemical and immunological properties similar to those of intact TnI. It binds TnC at a molar ratio of 1:1 and forms a ternary complex with TnC and TnT. TnC enhances the immunological activity of TnI153, but has little effect on the activity of TnI88. The TnI153-TnC complex exhibits higher immunological activity than rTnI-TnC and TnI88-TnC, and much higher activity than free rTnI, TnI153 and TnI88. The presence of TnT has no effect on the immunological activity of the TnI153-TnC complex, suggesting that the addition of TnT does not interfere with TnI153 recognition by TnI monoclonal antibodies. Free TnI153 and TnI88 degrade rapidly in human serum. TnC protects TnI153 from proteolytic degradation, but offers less protection for TnI88. The TnI88-TnC complex lost 80% of its immunological activity after incubation for 2 days in human serum at 37 degrees C. However, there was no loss in the immunological activity of the TnI153-TnC complex under the same conditions. A cTnI fragment (TnI80, residues 1-80), expressed in E. coli as a fusion protein, exhibits immunological activity and stability similar to that of TnI88.

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

BackgroundThe most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.ResultsWe developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.ConclusionsOur data demonstrate that the region surrounding the cysteines 238 and 243 in the α1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Mass Spectrometry of the Subunits of Highly Active Cytochrome b6f Complex from Spinach and the Cyanobacterium, M. laminosus: Identification of PetA,B,C,D,G,L,M,N Subunits in both Complexes and FNR in the Spinach b6f Complex

Highly active cytochrome b6f complexes from spinach and the cyanobacterium M. laminosus have been analyzed by liquid-chromatography with electrospray-ionization mass spectrometry (LC-MS). Both size-exclusion and reverse-phase separations were used to separate proteins from other contaminants allowing measurement of protein molecular weights to an accuracy exceeding 0.01 % (+/- 3Da at 30000 Da). Four small subunits corresponding to the products of petG, petL, petM and petN were detected in both complexes. The M. laminosus complex had four larger subunits corresponding to the products of petA, petB, petC and petD, a total of eight subunits. The spinach complex had the same four large subunits and a fifth polypeptide of molecular weight 35314.0 Da, a total of nine subunits. Based upon the mass of CNBr fragments found in a fraction collected simultaneously to the elution of this component we identified the fifth polypeptide as ferredoxin-NADP oxidoreductase (FNR). This is the largest number and range of integral membrane subunits determined by MS techniques in an integral membrane protein. The excellent resolution afforded by ESI-MS revealed profiles that define the native covalent state of the components of the complex as well as their molecular heterogeneity. Supported by NIH AI-12601-24 (JW), NIH GM-18457 (WAC), and the WM Keck Foundation.

Comparison of different electrokinetic separation modes applicable to a model peptide mixture (collagen type I and III CNBr fragments)

A number of electromigration separation modes were applied to the separation of CNBr-released peptides from rat tail tendon collagen (microemulsion electrokinetic chromatography, methanol- or ethanol-modified background electrolytes and the separation in the presence of molecular sieving effect exerting polymer, both in the presence and absence of SDS). Electrodriven separations with a Hypersil C8 packed capillary were investigated as well. The best separations were obtained with either the molecular sieving effect exerting polymer (polyethylene oxide) in the background electrolyte (whether SDS was present or absent) or with the electrodriven chromatography using the C8 reversed-phase packed capillary. In the latter separation system, it was possible to separate 25–27 peaks of the theoretically expected 24 peptides in the analyzed mixture of which 17 were at least tentatively identified. The additional peaks apparently stem from the incomplete cleavage of the parent collagen α chains. Successful separations can be done either with predominating molecular sieving or hydrophobic partitioning mechanism.