Antibodies against protein antigenic sites that are identical in the homologous protein of the immunized animal. Autoreactivity in rabbits of antibodies to sperm-whale myoglobin.

Sequence comparisons between the antigenic sites of sperm-whale myoglobin and the corresponding regions in rabbit myoglobin indicate that rabbits make antibodies to regions of the sperm-whale myoglobin molecule which are identical to the corresponding regions in rabbit myoglobin. Rabbit myoglobin did not precipitate with antisera to sperm-whale myoglobin. However, it exhibited an extensive cross-reaction as demonstrated by its ability to inhibit the precipitin reaction of sperm-whale myoglobin, and on an immunoadsorbent, bound a large amount of antibodies to sperm-whale myoglobin.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R _g ∼1.48–1.51 nm, helicity ∼75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0–3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2–11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations.     

Myoglobin as an Inhibitor of Exopeptidases from Lactobacillus sake

The effects of myoglobin on exopeptidases of Lactobacillus sake were determined. Inhibition of the aminopeptidases increased as the myoglobin concentration increased; aminopeptidase 3 was the most affected (90% inhibition). Aminopeptidases 1, 2, and 4 showed similar inhibition levels (around 60%). Myoglobin did not affect tripeptidase activity. Thus, myoglobin could limit amino acid generation in meat systems.     

3,3′,4,5′-Tetramethoxy-trans-stilbene Improves Insulin Resistance by Activating the IRS/PI3K/Akt Pathway and Inhibiting Oxidative Stress

The potential anti-diabetic effect of resveratrol derivative, 3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and its underlying mechanism in high glucose (HG) and dexamethasone (DXMS)-stimulated insulin-resistant HepG2 cells (IR-HepG2) were investigated. 3,3′,4,5′-TMS did not reduce the cell viability of IR-HepG2 cells at the concentrations of 0.5–10 µM. 3,3′,4,5′-TMS increased the potential of glucose consumption and glycogen synthesis in a concentration-dependent manner in IR-HepG2 cells. 3,3′,4,5′-TMS ameliorated insulin resistance by enhancing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), inhibiting phosphorylation of insulin receptor substrate-1 (IRS-1), and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells. Furthermore, 3,3′,4,5′-TMS significantly suppressed levels of reactive oxygen species (ROS) with up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. To conclude, the beneficial effect of 3,3′,4,5′-TMS against insulin resistance to increase glucose consumption and glycogen synthesis was mediated through activation of IRS/PI3K/Akt signaling pathways in the IR-HepG2 cells, accomplished with anti-oxidative activity through up-regulation of Nrf2.     

The binding of acetic anhydride- and citraconic anhydride-modified human low-density lipoprotein to mouse peritoneal macrophages The evidence for separate binding sites

Human plasma low-density lipoprotein (LDL) was modified chemically with either the monocarboxylic acid derivative, acetic anhydride, or the dicarboxylic acid derivative, citraconic anhydride, reagents which react principally with the lysine residues of protein. The modifications increased the net negative charge on the LDL particles, with citraconyl-LDL displaying a greater negative charge than acetylated LDL. Neither the antigenic reactivity nor the overall gross protein/lipid composition of the LDL were affected by the modification procedures, although a small reduction in the total cholesterol content was observed. The altered LDL species lost the ability to bind to the high-affinity cell surface B/E receptor but both bound to mouse peritoneal macrophages with saturable high-affinity kinetics. At 4°C, the macrophages bound ¹²⁵I-labelled citraconyl-LDL more avidly (K = 21 · 10⁻³ ml/ng) than they bound labelled acetyl-LDL(K = 2 · 10⁻³ ml/ng). Competitive inhibition studies indicated that acetyl-LDL and citraconyl-LDL were bound to non-identical sites on the macrophage monolayer surface and that the binding site for citraconyl-LDL was also different from that recognized by hypercholesterolaemic rabbit plasma VLDL (βVLDL).     

Observation of small conformational changes in the sperm-whale myoglobin by the far-infrared spectra

Small conformational changes in a molecule of sperm-whale myoglobin in its native solid state for different pH values at room temperature as well as during heat denaturation in alkali medium at different stages of unfolding of the globule were observed by using far-infrared spectroscopy in the region from 30 to 600 cm^?1. The changes appeared in the absorption bands near 420 and 470 cm^?1 ascribed to the side-chain vibrations of helical segments of the myoglobin molecule. For the first time the high structural sensitivity of the far-infrared region of the skeletal vibrations has been confirmed experimentally and the applicability of this technique to globular proteins demonstrated.     

Studies on myoglobin from the finback whale (Balaenoptera physalus). Preparation, physicochemical and immunochemical characterization, differentiation from sperm-whale myoglobin, amino acid composition and end-terminal analyses

1. Crystalline myoglobin was isolated from the skeletal muscle of the finback whale and fractionated, in its cyanmet form, into nine components (I-IX) by chromatography on CM-cellulose. Also in the cyanmet form, it was resolved into six components by electrophoresis on starch gel. Correspondence between the electrophoretic and chromatographic components was determined, and interconversion between components revealed by chromatography and electrophoresis. 2. The chromatographic myoglobin components were homogeneous in the ultra-centrifuge. Molecular weights of certain components were determined by means of sedimentation equilibrium and by gel filtration on Sephadex G-100. Values from these two methods corresponded to the minimum molecular weight calculated from the iron content. 3. The spectral properties of the chromatographic components were investigated in the visible and the ultraviolet ranges. 4. The major components of finback-whale myoglobin and sperm-whale myoglobin showed almost identical spectral, electrophoretic and chromatographic behaviours, but had different infrared spectra. The infrared spectra of the corresponding apoproteins were almost identical. 5. Rabbit antisera to sperm-whale myoglobin component X cross-reacted with finback-whale myoglobin components V, VI and VII only about 30%. 6. The major chromatographic components of finback-whale myoglobin have identical amino acid compositions. The polypeptide chain contains 151 amino acid residues and its molecular weight is 17504. 7. The N-terminal end of the chain is: [Formula: see text] Amino acids released from myoglobin by the action of carboxypeptidase A at different intervals were determined.     

Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

Analysis of 5-methoxytryptamine at the femtomole level in the rat and quail brain by gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry

A sensitive method for the measurement of endogenous 5-methoxytryptamine in brain tissue has been developed using capillary column gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry. 5-Methoxytryptamine was first converted to N-[²H₃]acetyl-5-methoxytryptamine by reaction with hexa-deuterated acetic anhydride, followed by reaction with pentafluoropropionic anhydride to yield the highly electron-capturing 3,3′-spirocyclic pentafluoropropionyl indolenine derivative. Quantitative analysis was carried out by selected-ion monitoring of the [M-HF] and [M-HF-DF] ion intensity of the 3,3′-spirocyclic pentafluoropropionyl indolenine derivative, using 5-methoxy-[α,α,β,β-²H₄]tryptamine as the internal standard. The presence of 5-methoxytryptamine in the brain tissue was demonstrated. In the absence of a monoamine oxidase inhibitor, the mean±S.D. levels of 5-methoxytryptamine in the rat and quail whole brain were found to be 30±6 and 347±52 pg/g, respectively. The possible physiological functions of 5-methoxytryptamine as a neuromodulator and/or neurotransmitter have to be considered.     

Perturbation of Human Coronary Artery Endothelial Cell Redox State and NADPH Generation by Methylglyoxal

Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100–500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of N ^ε-(carboxyethyl)lysine (CEL) and N ^ε-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.     

Haemoglobin adducts of aromatic amines: diamines and polyaromatic amines

Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[²H₈]aminofluoranthene, 3,3′-dimethyl-[²H₄]benzidine, [²H₄]benzidine, N′-acetyl-[²H₄]benzidine, 2,4-[²H₆]toluenediamine, 2,6-[²H₆]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC–MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine, 3,3′-dimethylbenzidine, 3,3′-methylenedianiline, 4,4′-methylenedianiline, N′-acetyl-benzidine, N′-acetyl-4,4′-methylenedianiline, 4,4′-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine.     

Increased 8-hydroxyguanine content of chloroplast DNA from ozone-treated plants

The mechanism of ozone-mediated plant injury is not known but has been postulated to involve oxygen free radicals. Hydroxyl free radicals react with DNA causing formation of many products, one of which is 8-hydroxyguanine. By using high performance liquid chromatography with electrochemical detection, the 8-hydroxy-2′-deoxyguanosine (8-OHdG) content of a DNA enzymatic digest can be sensitively quantitated. Beans (Phaseolus vulgaris L.) and peas (Pisum sativum L.) were treated with an ozone regime that caused acute injury. Chloroplast DNA was obtained from plants harvested either immediately after ozone treatment or 24 hours later. Ozone-exposed plants in general had nearly two-fold higher levels of 8-OHdG as compared to control plants. In vitro treatment of DNA in buffer solution with ozone did not cause formation of 8-OHdG in DNA, even though ozone did react directly with the macromolecule per se. Exposure of isolated, illuminated chloroplasts to ozone caused nearly a seven-fold increase in the amount of 8-OHdG in the chloroplast DNA as compared to none-ozone-exposed chloroplasts. These results suggest that ozone exposure to plants causes formation of enhanced levels of oxygen free radicals, thus mediating formation of 8-OHdG in chloroplast DNA. The reaction of ozone with DNA per se did not cause formation of 8-OHdG. Therefore, it is the interaction of ozone with plant cells and isolated chloroplasts which mediates oxygen free radical formation.     

1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.

1H-n.m.r. and c.d. studies on sperm-whale myoglobin show that the c.d. signal in the Soret region is inversely and linearly related to the proportion of minor isomer present. An alternative method, 'pH jump', is described for inducing orientational disorder in sperm-whale myoglobin without recourse to reconstitution. 1H-n.m.r. studies on human haemoglobin A indicate little heterogeneity in freshly isolated haemoglobin A, but the effect is enhanced in freeze-dried Sigma haemoglobin A.     

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc₁ complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc₁ complex were measured. Mixtures of bc₁ complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dualwavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 13 derivative produced a diminution of the polypeptide of the bc₁ complex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c₁, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc₁ complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.     

Isolation of a heme crevice peptide from affinity-labeled horseradish peroxidase

Apo-horseradish peroxidase was affinity-labeled with the monosulfuric anhydride derivative of mesoheme. The stoichiometry of heme anhydride binding was 1.1 moles of the anhydride per mole of apo-peroxidase.Tryptic digestion of the affinity-labeled peroxidase yielded a major lysine peptide which corresponded in composition to peptides T8 and T9a in the sequence of horseradish peroxidase (Welinder, K. G., Eur. J. Biochem. 96: 483–502, 1979) which contained one mole of histidine (histidine 170) per mole of peptide.     

Cyclic Guanosine 3′,5′-Monophosphate in the Dimorphic Fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3′,5′-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3′,5′-monophosphate has upon cell morphology.     

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

The binding of acetic anhydride- and citraconic anhydride-modified human low-density lipoprotein to mouse peritoneal macrophages. The evidence for separate binding sites

Human plasma low-density lipoprotein (LDL) was modified chemically with either the monocarboxylic acid derivative, acetic anhydride, or the dicarboxylic acid derivative, citraconic anhydride, reagents which react principally with the lysine residues of protein. The modifications increased the net negative charge on the LDL particles, with citraconyl-LDL displaying a greater negative charge than acetylated LDL. Neither the antigenic reactivity nor the overall gross protein/lipid composition of the LDL were affected by the modification procedures, although a small reduction in the total cholesterol content was observed. The altered LDL species lost the ability to bind to the high-affinity cell surface B/E receptor but both bound to mouse peritoneal macrophages with saturable high-affinity kinetics. At 4 degrees C, the macrophages bound 125I-labelled citraconyl-LDL more avidly (K = 21 X 10(-3) ml/ng) than they bound labelled acetyl-LDL (K = 2 X 10(-3) ml/ng). Competitive inhibition studies indicated that acetyl-LDL and citraconyl-LDL were bound to non-identical sites on the macrophage monolayer surface and that the binding site for citraconyl-LDL was also different from that recognized by hypercholesterolaemic rabbit plasma VLDL (beta VLDL).