Effect of ionic concentration on the higher-order structure of prophenol oxidase in Drosophila melanogaster

Phenol oxidase in Drosophila melanogaster occurs as precursors designated prophenol oxidases A₁ and A₃. Crossing experiments between isozyme variants proved that prophenol oxidase in this species is a homodimer. Prophenol oxidases were partially purified using ammonium sulfate fractionation, phenyl Sepharose, and DEAE-cellulose column chromatography. The preparations were mixed, then dialyzed against buffer containing varying salt concentrations. The resulting prophenol oxidase was analyzed by gel electrophoresis. At 20 mM KCl or NaCl, two bands of phenol oxidase were observed, corresponding to the parental ones as monomer, whereas at 200 mM KCl or NaCl, three bands appeared in the gel, one being a dimer. The monomer–dimer reversibility of the Drosophila prophenol oxidase depends on the salt concentrations. The phenol oxidase activity remained unaffected within the KCl concentrations tested. Considering the ionic concentration of Drosophila hemolymph, these results indicate that prophenol oxidase exists as a dimer in vivo, and the higher-order structure of prophenol oxidase can be altered reversibly by ionic concentrations in vitro.     

Prophenol oxidase A3 in Drosophila melanogaster: activation and the PCR-based cDNA sequence

Phenol oxidase exists in Drosophila hemolymph as a prophenol oxidase, A₁ and A₃, that is activated in vivo with a native activating system, AMM-1, by limited proteolysis with time. The polypeptide in purified prophenol oxidase A₃ has a molecular weight of approximately 77,000 Da. A PCR-based cDNA sequence coding A₃ has 2501 bp encoding an open reading frame of 682 amino acid residues. The potential copper-binding sites, from Trp-196 to Tyr-245, and from Asn-366 to Phe-421, are highly homologous to the corresponding sites in other invertebrates. The availability of prophenol oxidase cDNA should be useful in revealing the biochemical differences between A₁ and A₃ isoforms in Drosophila melanogaster that are refractory or unable to activate prophenol oxidase.     

Thermolabile Variant, PHOX-S, of Prophenol Oxidase in Drosophila melanogaster

The Phox(S) strain of Drosophila melanogaster is an electrophoretically slow variant found in a wild population at Victoria, Australia. Prophenol oxidase isoform A(1) from PHOX-S was purified and characterized biochemically and genetically. The purified fraction of A(1) from PHOX-S showed a homodimer with a molecular weight of the subunit of approximately 77 kDa. The Phox(S) strain was temperature sensitive in vivo in culture, and the purified protein was thermolabile in vitro. By the deletion mapping method, the Phox(S) locus was cytologically estimated to be at the location 55-A on the right arm of the second chromosome and 79.6 genetically. These data show that PHOX-S is an electrophoretic variant of MOX and that PHOX-S is the first thermolabile protein found in invertebrate prophenol oxidase.     

Use of nitrocellulose membrane to activate and measure insect prophenol oxidase

A method for the activation and measurement of insect prophenol oxidase using nitrocellulose membrane is presented. Using this method we were able to conveniently activate both crude and purified prophenol oxidase from insects belonging to three different orders. This rapid method allows for prophenol oxidase activation, in the absence of a prophenol oxidase-activating system, and in the presence of high ionic strength, protease inhibitors, or chelator.     

A link between blood coagulation and prophenol oxidase activation in arthropod host defense

Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade.     

Association of heat-induced conformational change with activity loss of Rubisco.

Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45 degrees C, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60 degrees C, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60 degrees C, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45 degrees C, partly reversible between 45 and 60 degrees C, and irreversible beyond 60 degrees C.     

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.     

Temperature-dependent beta-sheet formation in beta-amyloid Abeta(1-40) peptide in water: uncoupling beta-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimer's beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.     

Conformational study of human serum albumin in pre-denaturation temperatures by differential scanning calorimetry, circular dichroism and UV spectroscopy

Thermal conformational changes of human serum albumin (HSA) in phosphate buffer, 10 mM at pH = 7 are investigated using differential scanning calorimetric (DSC), circular dichroism (CD) and UV spectroscopic methods. The results indicate that temperature increment from 25 degrees C to 55 degrees C induces reversible conformational changes in the structure of HSA. Conformational change of HSA are shown to be a three-step process. Interestingly, melting temperature of the last domain is equal to the maximum value of fever in pathological conditions, i.e. 42 degrees C. These conformational alterations are accompanied by a mild alteration of secondary structures. Study of HSA-SDS (sodium dodecyl sulphate) interaction at 45 degrees C and 35 degrees C reveals that SDS affects the HSA structure at least in three steps: the first two steps result in more stabilization and compactness of HSA structure, while the last one induces the unfolding of HSA. Since HSA has a more affinity for SDS at 45 degrees C compared to 35 degrees C, It is suggested that the net negative charge of HSA is decreased in fever, which results in the decrease of HSA-associated cations and plasma osmolarity, and consequently, heat removal via the increase in urine volume.     

Reversible and irreversible coiled coils in the stalk domain of ncd motor protein

Ncd is a microtubule minus end-directed motor protein from Drosophila, a member of the kinesin-14 family, and an essential protein in mitosis and meiosis. Full-length ncd exists as a dimer via the formation of an alpha-helical coiled coil in its central stalk domain (P192-R346), which is thought to be one of the key regions for its motility. In our previous studies, however, none of the various synthetic polypeptide fragments (up to 46 residues) from the stalk domain formed a coiled coil. Herein, we have investigated the structural properties of the full-length ncd stalk domain using recombinant polypeptides together with shorter segments. These new fragments did form coiled coils as verified by far-UV circular dichroism (CD) spectroscopy and analytical ultracentrifugation, suggesting that a certain length of polypeptide would be required for dimer formation. Moreover, deletion mapping revealed that the cooperativity among the neighboring subdomains in the stalk domain is required for formation of the coiled coil. Interestingly, the intact stalk domain segments showed three-state transition in thermal unfolding measurements with CD, indicating the presence of two regions: (i) a coiled-coil region (P227-R306) that exhibits reversible denaturation at a lower temperature (20-30 degrees C) and (ii) a more rigid coiled-coil region (T307-E334) that exhibits irreversible denaturation at a high temperature (ca. 60 degrees C). These results imply that the N-terminal region of the stalk domain might be able to adopt both a coiled-coil conformation and a dissociated one, which might be relevant to the functions of ncd.     

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.     

High and low temperatures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts

We investigated the vertical transmission, reproductive phenotype, and infection density of a male-killing Spiroplasma symbiont in two Drosophila species under physiological high and low temperatures through successive host generations. In both the native host Drosophila nebulosa and the nonnative host Drosophila melanogaster, the symbiont infection and the male-killing phenotype were stably maintained at 25 degrees C, rapidly lost at 18 degrees C, and gradually lost at 28 degrees C. In the nonnative host, both the high and low temperatures significantly suppressed the infection density of the spiroplasma. In the native host, by contrast, the low temperature suppressed the infection density of the spiroplasma whereas the high temperature had little effect on the infection density. These results suggested that the low temperature suppresses both the infection density and the vertical transmission of the spiroplasma whereas the high temperature suppresses the vertical transmission preferentially. The spiroplasma density was consistently higher in the native host than in the nonnative host, suggesting that the host genotype may affect the infection density of the symbiont. The temperature- and genotype-dependent instability of the symbiont infection highlights a complex genotype-by-genotype-by-environment interaction and may be relevant to the low infection frequencies of the male-killing spiroplasmas in natural Drosophila populations.     

Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.     

Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus.

L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C. To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange. In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP. The results obtained from these measurements at various temperatures can be summarized as follows. No changes in the secondary-structure distribution are detectable from far-UV CD measurements. On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur. With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C. Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature. It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme. We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C. The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature on structure and functional properties of horseradish peroxidase

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.     

Temperature and the formation of radiation-induced chromosome aberrations. II. The temperature dependence of lesion repair and lesion interaction

Dicentric chromosome aberration yields have been measured after single-exposure and split-dose irradiations of human lymphocytes with 150 kV X-rays. Various temperature programmes between 4 and 37 degrees C were applied before, during and after irradiations and in the radiation-free interval. It was found that chromatin lesion repair was completely suppressed at 21 degrees C and below, whereas lesion formation is reduced only below 17 degrees C. The interaction between repairable lesions which leads to exchange-type aberrations is also suppressed by low temperatures. Hypothermic suppression of chromatin lesion repair and interaction is fully reversible at least up to 12 h of maintenance of the 'stored' state of these lesions.     

Expression,purification, and characterization of pro‐phenoloxidase‐activating serine protease from Spodoptera litura

One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6 h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni‐NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N‐terminal sequencing of processed phenol oxidase revealed 11 kDa propeptide instead of in‐silico predicted 6 kDa polypeptide. © 2009 Wiley Periodicals, Inc.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.