Ruthenium-modified proteins. Reactions of cis-[ Ru(NH3)4(OH2)2]2+ and cis-[ Ru(en)2(OH2)2]2+ with azurin,myoglobin and cytochrome c

Reactions of cis-[Ru(en)₂(OH₂)₂]²⁺ (or cis-[Ru (NH₃)₄(OH₂)₂]²⁺) with Pseudomonas aeruginosa azurin (Az), horse heart myoglobin (Mb^h), and horse heart cytochrome c (cyt c) give Ru-labelled proteins. The ruthenium binding sites in the singly modified derivatives are His-83 (Az), His-81 (Mb^h), and His-33 (cyt c). Spectroscopic and electrochemical measurements indicate that the structures of the proteins are not perturbed by the surface-bound ruthenium complexes. The E°_f values of the Ru(III)/(II) couple in these Ru-modified proteins fall between −0.07 and −0.13 V vs. NHE.     

Comparative Studies of the Indoleamine Dioxygenase-like Myoglobin from the Abalone Sulculus diversicolor

The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass. The myoglobin is homodimeric and the molecular mass of the constituent polypeptide chain is 41,000 Da. The amino acid sequence and gene structure are highly homologous with those of a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO). Thus Sulculus myoglobin evolved from an IDO gene, and represents a typical case of functional convergence. The oxygen equilibrium properties of Sulculus myoglobin were examined and compared with those of myoglobins from other sources. It binds oxygen reversibly, and the P₅₀ was determined to be 3.8 mmHg at 20°C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is significantly lower than those of usual 16 kDa myoglobins. It also displays no cooperativity (n_max: 1.02–1.06) and no alkaline Bohr effect between pH 7.0 and 7.9. The cDNA-derived amino acid sequences of vertebrate IDOs, molluscan IDO-like myoglobins and a homolog in the yeast Saccharomyces were aligned, and several amino acid residues were proposed as candidates for key residues to control the function of IDO or myoglobin.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Synthesis and Antitumor Activity of Acyl and Benzyl Types of Prodrugs of 2′-Deoxy-2′-methylidenecytidine

Abstract

For the purpose of improvement of the in vivo antitumor activity of 2′-deoxy-2′-methylidenecytidine (DMDC, 1), we synthesized its various acyl and benzyl derivatives and evaluated them for their antitumor activity against P388 murine leukemia in mice. In terms of minimum effective dose (30% increase in life span), 5′-O-stearoyl DMDC showed two-fold higher antitumor activity than DMDC on a molar basis, when intraperitoneally (i.p.) administered to mice once a day. The antitumor activities of some other acyl derivatives were almost comparable to that of DMDC, while benzyl derivatives had no antitumor activity. Results on the hydrolysis of 5′-O-acyl derivatives by porcine liver esterase showed that at least these derivatives should not be resistant to enzymatic hydrolysis for exhibiting antitumor activity. After either an i.p. or oral dose of 3′-O-benzyl DMDC, very low concentrations of blood DMDC were seen compared with those after administration of DMDC, suggesting that the inactivity of benzyl derivatives as prodrugs was due to the minimal level of DMDC in circulation after administration.     

Structure-Activity Relationships Amongst β-Lactamase Inhibitors

Abstract

Using a variety of β-lactamases including those from Escherichia coli (TEM-1), Enterobacter cloacae P99 and Staphylococcus aureus the inhibition profiles (I₅₀ values) were determined for various groups of compounds including penicillins, penicillanic acid derivatives (sulphone and β-halo substitutions), olivanic acids and clavulanic acid derivatives including substituted ethers and amines. Some of the latter compounds had higher activity than clavulanic acid with and without preincubation of enzyme with inhibitor but they still had poor activity against the P99 enzyme. Improvements in activity against Class I cephalosporinases were obtained with some derivatives of clavulanic acid but this was usually achieved at the expense of activity against clavulanate susceptible β-lactamases.The olivanic acids had the highest activity against the widest range of β-lactamases.     

Amino Acid Sequence, Spectral, Oxygen-Binding, and Autoxidation Properties of Indoleamine Dioxygenase-Like Myoglobin from the Gastropod Mollusc Turbo cornutus

Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS–PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of to CT maximum (/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P ₅₀ = 3.5 mm Hg at pH 7.4 and 20°C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pK _a = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pK _a 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.     

Myoglobin with modified tetrapyrrole chromophores: Binding specificity and photochemistry

Complexes were prepared of horse heart myoglobin with derivatives of (bacterio)chlorophylls and the linear tetrapyrrole, phycocyanobilin. Structural factors important for binding are (i) the presence of a central metal with open ligation site, which even induces binding of phycocyanobilin, and (ii) the absence of the hydrophobic esterifying alcohol, phytol. Binding is further modulated by the stereochemistry at the isocyclic ring. The binding pocket can act as a reaction chamber: with enolizable substrates, apo-myoglobin acts as a 13²-epimerase converting, e.g., Zn-pheophorbide a' (13²S) to a (13²R). Light-induced reduction and oxidation of the bound pigments are accelerated as compared to solution. Some flexibility of the myoglobin is required for these reactions to occur; a nucleophile is required near the chromophores for photoreduction (Krasnovskii reaction), and oxygen for photooxidation. Oxidation of the bacteriochlorin in the complex and in aqueous solution continues in the dark.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.     

Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

Interaction of porcine circovirus type 2 replication with intracellular redox status in vitro

Abstract

Objectives

Redox status influences replication of some viruses but its effect on porcine circovirus type 2 (PCV2), the primary causative agent of the emerging swine disease post-weaning multisystemic wasting syndrome is not known. The interaction of PCV2 replication with intracellular redox status in PK15 cells was examined in this study.

Methods

Intracellular glutathione (GSH) was measured spectrophotometrically by reaction with 5, 5′-dithiobis (2-nitrobenzoic acid). Total superoxide dismutase activity (SOD) was assayed by inhibition of oxyamine oxidation by the xanthine oxidase system. Malondialdehyde (MDA) was assayed spectrophotometrically using the thiobarbituric acid reaction. Both quantification of PCV2 DNA by real-time polymerase chain reaction and indirect immunofluorescence of PCV2-infected cells were used to evaluate the replication of PCV2.

Results

Both GSH and SOD decreased significantly at 48 hours after PCV2 infection, whereas MDA concentration increased significantly after 48 hour post-infection. Furthermore, PCV2 replication in PK15 cells was significantly impaired after the elevation of intracellular GSH through treatment with the antioxidant N-acetyl-l-cysteine (NAC), a precursor in GSH synthesis. In contrast, PCV2 replication in PK15 cells was enhanced after reduction of GSH levels through H₂O₂-mediated oxidation. In addition, NAC treatment blocked the increase of virus replication induced by H₂O₂.

Conclusions

This study suggests that PCV2 infection induces oxidative stress and that intracellular redox status influences PCV2 replication in PK15 cells.     

Synthesis and Antiviral Evaluation of N-β-D-Ribosides of Ergot Alkaloids

Abstract

N-β-D-Ribosides of agroclavine (1), elymoclavine (2), lysergene (4), lysergol (3), and 9, 10-dihydrolysergol (5) were prepared by SnCl₄ catalyzed ribosylation of their TMS derivatives with 1-O-acetyl-2, 3,5-tri-O-benzoyl-β-D-ribofuranose. None of the new compounds exhibited activity against HIV or other viruses tested.     

Antioxidant activity of aminodiarylamines in the thieno[3,2-b]pyridine series: radical scavenging activity,lipid peroxidation inhibition and redox profile

Abstract

The antioxidant activity of the aminodi(hetero)arylamines, prepared by C–N coupling of the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate with bromonitrobenzenes and further reduction of the obtained nitro compounds, was evaluated by chemical, biochemical and electrochemical assays. The aminodi(hetero)arylamine with the amino group ortho to the NH and a methoxy group in para, was the most efficient in radical scavenging activity (RSA, 63?µM) and reducing power (RP, 33?µM), while the aminodiarylamine with the amino group in para to the NH, gave the best results in β-carotene-linoleate system (41?µM) and inhibition of formation of thiobarbituric acid reactive substances in porcine brain cells homogenates (7?µM), with EC₅₀ values even lower than those obtained for the standard trolox. This diarylamine also presented the lowest oxidation potential, lower than the one of trolox, and the highest antioxidant power in the electrochemical assays. The para substitution with an amino group enables higher antioxidant potential.     

Ribose sugars generate internal glycation cross-links in horse heart myoglobin

Glycation of horse heart metmyoglobin with d-ribose 5-phosphate (R5P), d-2-deoxyribose 5-phosphate (dR5P), and d-ribose with inorganic phosphate at 37 °C generates an altered protein (Myo-X) with increased SDS–PAGE mobility. The novel protein product has been observed only for reactions with the protein myoglobin and it is not evident with other common sugars reacted over a 1 week period. Myo-X is first observed at 1–2 days at 37 °C along with a second form that is consistent in mass with that of myoglobin attached to several sugars. MALDI mass spectrometry and other techniques show no evidence of the cleavage of a peptide from the myoglobin chain. Apomyoglobin in reaction with R5P also exhibited this protein form suggesting its occurrence was not heme-related. While significant amounts of O₂⁻ and H₂O₂ are generated during the R5P glycation reaction, they do not appear to play roles in the formation of the new form. The modification is likely due to an internal cross-link formed during a glycation reaction involving the N-terminus and an internal amine group; most likely the neighboring Lys133. The study shows the unique nature of these common pentose sugars in spontaneous glycation reactions with proteins.     

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c₅₅₁ (PAc₅₅₁) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc₅₅₁ denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc₅₅₁ denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc₅₅₁, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.     

The role of entropy in the discrimination between CO and O2 in myoglobin

Using stopped-flow rapid mixing and flash photolysis techniques, the dissociation rate coefficients of horse carbonmonoxy myoglobin (hMbCO) and oxygenated myoglobin (hMbO₂) in aqueous solution have been determined as a function of temperature between 274 and 342 K. From the Arrhenius plot, an activation enthalpy for dissociation of 74 kJ/mol was obtained for both ligands. The pronounced kinetic differences arise from markedly different pre-exponentials. We compare the Arrhenius parameters with those of the association reaction, as measured at cryogenic temperatures. In our analysis we conclude that the entropy loss upon binding of O₂ is twice as large as that for CO. Taking reasonable estimates for the frequency factor, the transition state entropy in hMbO₂ is located roughly half way in between the entropies of the bound and unbound states. By contrast, the entropy of the transition state in hMbCO appears to be identical to that of the bound state. Possible structural reasons for the different behavior are discussed. Received: 13 January 1997 / Accepted: 24 April 1997     

Purification and Characterization of Class Alpha and Mu Glutathione S-Transferases From Porcine Liver

Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M_r = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M_r values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M_r = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.     

Expression and transcriptional activity of alternative splice variants of Mitf exon 6

Heme proteins––hemoglobin and myoglobin possess esterase activities. Studies with purified hemoglobin from normal individuals and diabetic patients revealed that the esterase activity as measured from hydrolysis of p-nitrophenyl acetate (p-NPA) was higher in diabetic condition and increased progressively with extent of the disease. HbA_1c, the major glycated hemoglobin, which increases proportionately with blood glucose level in diabetes mellitus, exhibited more esterase activity than the non-glycated hemoglobin fraction, HbA₀, as demonstrated spectrophotometrically as well as by activity staining. Glycation influenced esterase activity of hemoglobin by increasing the affinity for the substrate and the rate of the reaction. Both HbA₀ and HbA_1c-mediated catalysis of p-NPA hydrolysis was pH-dependent. Esterase activity of in vitro-glycated myoglobin (GMb) was also higher than that of its non-glycated analog (Mb). The amplified esterase activities of hemoglobin and myoglobin might be associated with glycation-induced structural modifications of the proteins.     

Sage-Grouse Population Dynamics are Adversely Affected by Overabundant Feral Horses

In recent decades, feral horse (Equus caballus; horse) populations increased in sagebrush (Artimesia spp.) ecosystems, especially within the Great Basin, to the point of exceeding maximum appropriate management levels (AML_max), which were set by land administrators to balance resource use by feral horses, livestock, and wildlife. Concomitantly, greater sage-grouse (Centrocercus urophasianus; sage-grouse) are sagebrush obligates that have experienced population declines within these same arid environments as a result of steady and continued loss of seasonal habitats. Although a strong body of research indicates that overabundant populations of horses degrade sagebrush ecosystems, empirical evidence linking horse abundance to sage-grouse population dynamics is missing. Within a Bayesian framework, we employed state-space models to estimate population rate of change (λ) using 15 years (2005–2019) of count surveys of male sage-grouse at traditional breeding grounds (i.e., leks) as a function of horse abundance relative to AML_max and other environmental covariates (e.g., wildfire, precipitation, % sagebrush cover). Additionally, we employed a post hoc impact-control design to validate existing AML_max values as related to sage-grouse population responses, and to help control for environmental stochasticity and broad-scale oscillations in sage-grouse abundance. On average, for every 50% increase in horse abundance over AML_max, our model predicted an annual decline in sage-grouse abundance by 2.6%. Horse abundance at or below AML_max coincided with sage-grouse λ estimates that were consistent with trends at non-horse areas elsewhere in the study region. Thus, AML_max, as a whole, appeared to be set adequately in preventing adverse effects to sage-grouse populations. Results indicated 76%, 97%, and >99% probability of sage-grouse population decline relative to controls when horse numbers are 2, 2.5, and ≥3 times over AML_max, respectively. As of 2019, horse herds exceeded AML_max in Nevada, USA, by >4 times on average across all horse management areas. If feral horse populations continue to grow at current rates unabated, model projections indicate sage-grouse populations will be reduced within horse-occupied areas by >70.0% by 2034 (15-year projection), on average compared to 21.2% estimated for control sites. A monitoring framework that improves on estimating horse abundance and identifying responses of sage-grouse and other key indicator species (plant and animal) would be beneficial to guide management decisions that promote co-occurrence of horses with sensitive wildlife and livestock within landscapes subjected to multiple uses. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)-NO) involves the transient ferric nitrosylated species (Mb(III)-NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)-O₂) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV)O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)-CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)-CO (i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)-CO (i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10⁻² s⁻¹ and k_off(CO) = (1.4 ± 0.2) × 10⁻² s⁻¹, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k_off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)-CO.