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1.
Pancreatic ribonuclease A (RNase A) has been shown to aggregate moderately and gradually at 65 degrees C. Antibodies raised against the dodecapeptide KETAAAKFERQG corresponding to the N-terminal 1-12 amino acid residues of RNase A (Npep) as well as native RNase A were effective in lowering RNase A aggregation at 65 degrees C. The antiRNase A antibodies were, however, more protective. The binding of antiNpep antibodies to the N-terminal region of RNase A may interfere with initiation of oligomerization of the enzyme and consequently its aggregation. The antiRNase A antibodies were presumably more effective in protecting RNase A against aggregation by binding to multiple epitopes of the enzyme including the N-terminal region and hence restricting the interaction of the monomers.  相似文献   

2.
The influence of glycosylation on proteolytic degradation was studied by comparing cleavage sites in ribonuclease A (RNase A) and ribonuclease B (RNase B), which only differ by a carbohydrate chain attached to Asn34 in RNase B. Primary cleavage sites in RNase B were determined by identifying complementary fragments using matrix-assisted laser desorption/ionization mass spectrometry and compared with those in RNase A [Arnold et al. (1996), Eur. J. Biochem. 237, 862–869]. RNase B was cleaved by subtilisin even at 25°C at Ala20–Ser21 as known for RNase A. Under thermal unfolding, the peptide bonds Asn34–Leu35 and Thr45–Phe46 were identified as primary cleavage sites for thermolysin and Lys31–Ser32 for trypsin. These sites are widely identical with those in RNase A. Treatment of reduced and carbamidomethylated RNase A and RNase B with trypsin led to a fast degradation and revealed new primary cleavage sites. Therefore, the state of unfolding seems to determine the sequence of degradation steps more than steric hindrance by the carbohydrate moiety does.  相似文献   

3.
The effect of temperature and O2 saturation on the production of recombinant proteins -galactosidase and human glucocerebrosidase by Spodoptera frugiperda cells (Sf9) infected with recombinant Autographa californica nuclear polyhedrosis virus was investigated. The rates of cell growth, glucose consumption, O2 consumption and product expression were measured at temperatures between 22° C and 35° C. The results indicated that possible O2 limitation may be alleviated without compromising the maximum cell yield by lowering the incubation temperature from 27° C to 25° C. The expression level of the recombinant proteins at 27° C was similar to that obtained at 22° C and 25° C; lower protein yields were obtained at 30° C. An increase in temperature from 22° C to 27° C led to earlier production of the proteins and to an increase in the proportion of the product released outside the cells. Correspondence to: J. Shiloach  相似文献   

4.
Summary Values for basal metabolism, standard tidal volume (V T), standard minute volume ( ), and mean extraction efficiency (EO2) in the thermal neutral zone (TNZ) inAgapornis roseicollis (1.84 ml·min–1; 0.95 ml·br–1, STPD; and 33.3 ml·min–1, STPD; and 22.5%; respectively) were all very similar to values for these parameters previously measured inBolborhynchus lineola, a similarly sized, closely related species from a distinctly different habitat.Having both a lower critical temperature (Tlc) below and an upper critical temperature (Tuc) above those ofB. lineola, the TNZ ofA. roseicollis extended from 25° to at least 35°C. The thermal conductance below the TNZ ofA. roseicollis was 14% less than that ofB. lineola. Therefore, at 5°C the standard metabolic rate (SMR) of the former is 17% less than that of the latter, and at 35°C it is 20% less. At 5°CA. roseicollis has a lower EO2 and at 35°C a higher EO2 than that ofB. lineola. The patterns of resting energy metabolism and of ventilation ofA. roseicollis and ofB. lineola are consistent with the former species being better suited to living in a more variable thermal environment than the latter.MeanV T has a weak positive correlation with the rate of oxygen consumption ( ) at a constant ambient temperature (T a) but a much stronger correlation when resting increases in response to a decrease inT a.V t is the only ventilatory parameter which is linearly correlated toT a from 35° to –25°C. The data suggest thatT a may have a regulatory effect onV T somewhat independent of or .  相似文献   

5.
Development, survival and reproduction of Euseius finlandicus Oudemans were studied at seven constant temperatures (15, 20, 25, 27, 30, 32 and 34°C) in the laboratory. Within the temperature range tested, developmental period from egg to adult varied from 148 to 360.5h and 133.7 to 336.5h for females and males, respectively. The lower thermal threshold for immature development for females and males was 8.9 and 6.4°C, respectively. Survival during immature development exceeded 90% at all the temperatures from 15 to 32°C, but at 34°C an abrupt decline was recorded. Female longevity decreased gradually from 82.7d at 15°C to 12.2 d at 34°C. The mean generation time ranged from 44.3d at 15°C to 15.9d at 32°C. The highest r m value (0.2817) was obtained at 30°C and the lowest at 15°C (0.0976). Temperatures above 30°C had an adverse effect on population increase.  相似文献   

6.
Summary To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [3H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37°C and 45°C were similarly stable. At 37°C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45°C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37°C. Shift back to 37°C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue - methylene ATP during 45°C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45°C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or Pi had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).  相似文献   

7.
Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm2, 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm2, 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k in, total inactivation; k in *, thermal inactivation; and k in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).  相似文献   

8.
The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]0. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]0 (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]0 and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A lim; t} (t is time and A lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.  相似文献   

9.
A theoretical model is presented to study the stepwise thermal unfolding of globular proteins using the stabilizing/destabilizing characters of amino acid residues in protein crystals. A multiple regression relation connecting the melting temperature and the amounts of stabilizing and destabilizing groups of residues in a protein, when used for the thermal behavior of peptide segments, provides reliable results on the stepwise unfolding nature of the protein. In ribonuclease A, the shell residues 16–22 are predicted to unfold earlier in the temperature range 30–45°C; the -sheet structures undergo thermal denaturation as a single cooperative unit and there is evidence indicating the segment 106–118 as a nucleation site. In ribonuclease S, the S-peptide unfolds earlier than S-protein. The predicted average and the range of melting temperatures, and the folding pathways of a set of globular proteins, agree very well with the experimental results. The results obtained in the present study indicate that (i) most of the nucleation parts possess high relative thermal stability, (ii) the unfolded state retains some residual structure, and (iii) some segments undergo gradual and overlapping thermal denaturation.  相似文献   

10.
Sharma PK  Kumar R  Kumar R  Mohammad O  Singh R  Kaur J 《Gene》2012,491(2):264-271
A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60 °C. Interestingly, the overall catalytic efficiency (kcat/Km) of mutant was also enhanced (~ 20 folds). Circular dichroism spectroscopy, studied as function of temperature, demonstrated that the mutant lipase retained its secondary structure up to 70-80 °C, whereas wild type protein structure was completely distorted above 35 °C. Additionally, the intrinsic tryptophan fluorescence (a probe for the tertiary structure) also displayed difference in the conformation of two enzymes during temperature dependent unfolding. Furthermore, mutation N355K resulted in extensive H-bonding (Lys355 HZ1OE2 Glu284) with a distance 2.44 Å. In contrast to this, Wt enzyme has not shown such H-bonding interaction.  相似文献   

11.
Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (AMG1) into another (IMG2).  相似文献   

12.
The thermal stability of glucose oxidase was studied at temperatures between 50 and 70°C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (<15 g/ml) and pH > 5.5, the rate constants k in, s–1, for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of structures in stabilizing the protein globule. At a concentration of 15 g protein/ml, the activation energy E Aof thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60°C had the following values: H= 78.4 kcal/mol, G= 25.5 kcal/mol, and S= 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.  相似文献   

13.
Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and afterin vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circulardichroism measurements and limited trypsin digestion. Melting of fibrils from standardizedin vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (T m ) at 43.3°C in 0.05% acetic acid. This is 1.9°C above control skin (T m =41.4°C), most likely, due to a higher degree of prolyl hydroxylation—0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce theT m slightly (1°C). Underhydroxylated bone collagen has aT m which is 2°C below control. Melting temperatures ofin vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3°C, exceeds control bone by 1.4°C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4°C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.  相似文献   

14.
Summary Oxygen consumption at 25°C was measured continously throughout the egg stage of Leptopterna dolobrata (more than 9 months).The rate of O2-uptake (l O2/100 eggs · 1 h) is low in freshly laid eggs. Maintaining the eggs at a constant temperature of 16°C, respiration rises abruptly from the first day after oviposition and continues rising steadily for 3 days, reaching an average value of 1.4 l. Oxygen consumption persists at or near this high level during the developmental phase of prediapause, which lasts about 15 days. After some days of oscillating high and low values, respiration decreases, and from the 24th day a low level (0.3–0.4 l) is reached. If the eggs are incubated at 16°C continuously, this low diapause-level is maintained until the end of the experiments (42 weeks) and diapause is terminated in a few eggs only.A significant increase in the success of hatching is obtained by exposing the eggs to a sufficient period of chilling.24 groups of diapausing eggs were chilled at 5°C for certain periods (10, 18, 22, 26, 31, and 34 weeks) and afterwards transferred to 16°C and re-incubated. The changes of their O2-uptake at 25°C were traced throughout their chilling and successive re-incubating periods.Oxygen consumption is greatly accelerated during the cold treatment of the eggs. The low values of the diapause-level are raised progressively during the first 6 weeks of chilling. After this primary rapid increase, respiration remains at a level 5-times as high as the diapause values over a period up to the 25th week at 5°C. This is almost exactly the duration of mesodiapause (6 months).The rates of O2-uptake during the subsequent re-incubation at 16°C depend on the extent of chilling. The ability of diapause-breaking is correlated to the rates of O2-uptake, measured after setting of re-incubation. If respiration never decreases by the onset of re-incubation, diapause is terminated in most of the eggs, and the rates of O2-uptake increase as re-incubation goes on towards the emergence of the larvae (postdiapause period).A preliminary interpretation of the cold-stimulated O2-uptake in diapausing Leptopterna-eggs is given.Dedicated to Prof. Dr. H. Precht (Kiel) on the occasion of his 65th birthday  相似文献   

15.
Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G D at 25 °C, is 59±5 kJ mol–1 (DSC) and 65±6 kJ mol–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode  相似文献   

16.
Summary The kinetic and stability characteristics of crude extract nitrile hydratase fromBrevibacterium R-312 were studied for the hydration of 3-cyanopyridine to nicotinamide. The enzyme was substrate and product inhibited and had the following kinetic constants:K m =28 mM;K p =36 mM;K s =155 mM;V m =5.8 mol/min/mg protein (25°C). Itsmaximum temperature and pH (phosphate buffer) were 35°C and 8.0, respectively and it had half-lives of 50 days, 10 days and 1 day at 4°C, 10°C and 25°C, respectively. The crude extract also exhibited amidase activity on nicotinamide, but it became significant only at nicotinamide concentrations greater than 300 mM. Mathematical models for batch and fed-batch hydrations were developed to account for substrate and product inhibitions and for enzyme decay. They predicted to within 10% experimental results for initial substrate and final product concentrations up to 300 mM; the accuracies decreased at higher concentrations primarily because of the relatively rapid hydrolysis of nicotinamide.  相似文献   

17.
Chitosan interaction with soybean β-conglycinin β3 was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T m) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T m value was elevated by 7.7 °C, whereas the T m elevation upon addition of chitosan hexamer (GlcN)6 was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T m elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T m elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.  相似文献   

18.
Effects of temperature on properties of flight neurons in the locust   总被引:1,自引:0,他引:1  
High ambient temperatures increase the wing-beat frequency in flying locusts, Locusta migratoria. We investigated parameters of circuit and cellular properties of flight motoneurons at temperatures permissive for flight (20–40 °C). As the thoracic temperature increased motoneuronal conduction velocity increased from an average of 4.40 m/s at 25 °C to 6.73 m/s at 35 °C, and the membrane time constant decreased from 11.45 ms to 7.52 ms. These property changes may increase locust wing-beat frequency by affecting the temporal summation of inputs to flight neurons in the central circuitry. Increases in thoracic temperature from 25–35 °C also resulted in a hyperpolarization of the resting membrane potentials of flight motoneurons from an average of-41.1 mV to -47.5 mV, and a decrease of input resistances from an average of 3.45 M to 2.00 M. Temperature affected the measured input resistance both by affecting membrane properties, and by altering synaptic input. We suggest that the increase in conduction velocity Q10=1.53) and the decrease of membrane time constant (Q10=0.62) would more than account for the wing-beat frequency increase (Q10=1.15). Hyperpolarization of the resting membrane potential (Q10=1.18) and reduction in input resistance (Q10=0.54) may be involved in automatic compensation of temperature effects.Abbreviations ANOVA analysis of variance - CPG central pattern generator - DL dorsal longitudinal muscles - EMG electromyographic - MN motoneuron - PSP post synaptic potential - Q10 temperature coefficient - RMP resting membrane potential - S.D. standard deviation - SR stretch receptor  相似文献   

19.
Summary Lactate concentration was measured in the abdominal muscle of the shrimpPalaemon serratus. Rapid and seasonal temperature changes result in an increase of the lactate content of approximately 3–4 fold.Lactate dehydrogenase from the abdominal muscle exhibits a temperature dependent pyruvate inhibition with pyruvate as substrate.The kinetic parameters of lactate dehydrogenase fromPalaemon serratus are found to vary during rapid temperature changes: Vmax increases with temperature from 0.06 mol min–1 (mg protein)–1 at 10°C to 0.28 mol min–1 (mg protein)–1 at 30°C with lactate as substrate, and from 5.5 mol min–1 (mg protein)–1 at 10°C to 26.2 mol min–1 (mg protein)–1 at 30°C, with pyruvate (Table 1). The Hill coefficientn H, decreases with temperature from 2.2 to 1.2 when the pyruvate reduction is examined, but remains near 1.2 when the activity is measured with lactate as substrate (Table 1). The S0.5 values for lactate show a tendency to increase below 30 °C (18.9 mM l–1 at 20 °C) whereas the S0.5 for pyruvate is found to increase greatly with temperature (0.004 mM l–1 at 10 °C and 0.06 mM l–1 at 20 °C).Long term temperature changes involve variations of lactate dehydrogenase activity leading to inverse thermal compensation (Table 2).Activation energy (about 56 kJ both with pyruvate and lactate) does not vary during the year, suggesting that temperature adaptation does not induce important catalytic changes (Table 3).Abbreviation LDH lactate dehydrogenase  相似文献   

20.
When individual mice were examined, it was found that the colonic body temperatureT col of each individual within a genetically heterogeneous population tended to remain either above (warm) or below (cool) the population mean.T col of warm, but not cool, mice showed circadian variation. When exposed to aT a of 43° C, theT col of cool mice increased by as musch as 2.4° C more than that of warm mice for a given 15 min increment of heating at 43°C. Survival of mice after acute lethal heat load (LD75, –45°C) was significantly inversely correlated withT col. Small persistent differences in body temperature of individuals may indicate differing thermal adaptedness.  相似文献   

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