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1.
Secretion of both partially unfolded and folded apoproteins of dimethyl sulfoxide reductase by spheroplasts from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans.
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Spheroplasts prepared from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans secreted dimethyl sulfoxide (DMSO) reductase which had no molybdenum cofactor and therefore no activity, whereas those from wild-type cells secreted the active reductase. The inactive DMSO reductase proteins were separated by nondenaturing electrophoresis into two forms: form I, with the same mobility as the native enzyme, and form II, with slower mobility. Both forms had the same mobility on denaturing gel. Form I and active DMSO reductase had the same profile on gel filtration chromatography. Form II was eluted a little faster than the native enzyme, suggesting that DMSO reductase form II was not an aggregated form but a compactly folded form very similar to the native enzyme. Form II was digested by trypsin and denatured with urea, whereas form I was unaffected, like native DMSO reductase. These results suggested that form II was a partially unfolded but compactly folded apoprotein of DMSO reductase. 相似文献
2.
A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme 总被引:2,自引:0,他引:2
Photochemically induced dynamic nuclear polarization (photo-CIDNP) techniques have been used to examine denatured states of lysozyme produced under a variety of conditions. 1H CIDNP difference spectra of lysozyme denatured thermally, by the addition of 10 M urea, or by the complete reduction of its four disulfide bonds were found to differ substantially not only from the spectrum of the native protein but also from that expected for a completely unstructured polypeptide chain. Specifically, denatured lysozyme showed a much reduced enhancement of tryptophan relative to tyrosine than did a mixture of blocked amino acids with the same composition as the intact protein. By contrast, the CIDNP spectrum of lysozyme denatured in dimethyl sulfoxide solution was found to be similar to that expected for a random coil. It is proposed that nonrandom hydrophobic interactions are present within the denatured states of lysozyme in aqueous solution and that these reduce the reactivity of tryptophan residues relative to tyrosine residues. Characterization of such interactions is likely to be of considerable significance for an understanding of the process of protein folding. 相似文献
3.
Thermal transitions were measured by differential scanning calorimetry for rabbit cardiac sarcolemma in 3-(N-morpholino)propanesulfonic acid buffer at pH 7.5, in glycerol-buffer and dimethyl sulfoxide - buffer mixtures, after heat denaturation, and after enzymatic degradation of the proteins. Specific solvent effects on the protein transitions were observed. Glycerol stabilized some of the four protein transitions, while dimethyl sulfoxide destabilized all protein transitions. The thermal transitions in the lower temperature range were studied for both the membranes and the lipid extracted from the membranes. A very small endotherm was observed for both the lipid extracted from the sarcolemma and the intact membrane (0.1-0.2 cal/g; 1 cal = 4.1868 J). A larger endotherm was observed in both the glycerol-buffer and dimethyl sulfoxide - buffer mixtures. Major perturbation of the protein by enzymatic degradation (papain or trypsin digestion), by heat denaturation, or by reaction with excess N-ethylmaleimide all produced larger endotherms near 20 degrees C. The very small magnitude of the endotherm near 20 degrees C suggests that it is not a typical gel - liquid crystalline transition of the bilayer. However, the occurrence of an endotherm in the extracted lipid suggests that some reorientation of lipid is involved. 相似文献
4.
Inactive denatured trypsin changes into active native trypsin in the protein solutions which have been used to estimate tryptic activity. If the digestion mixture, however, is alkaline enough and contains enough urea this change does not take place. Such a digestion mixture can be used to estimate active native trypsin in the presence of inactive denatured trypsin. 相似文献
5.
It is confirmed from sedimentation studies that only a small fraction of diguaninyl derivatives formed on reaction on reactions of native DNA with mustard gas crosslink the two DNA chains. It is also apparent that heavily alkylated DNA can be studied in the denatured form in dimethyl sulfoxide without the gross degradation that results if denaturation is effected by alkali. 相似文献
6.
Escherichia coli FtsH is a membrane-bound and ATP-dependent protease which degrades some soluble and integral membrane proteins. The N-terminal region of FtsH mediates membrane association as well as homooligomeric interaction of this enzyme. Previously, we studied in vivo functionality of FtsH derivatives, in which the N-terminal membrane region was either deleted (FtsH(DeltaTM)), replaced by a leucine zipper (Zip-FtsH(DeltaTM)), or replaced by a lactose permease transmembrane segment (LacY-FtsH). It was indicated that homooligomerization is required for the minimum proteolytic activity, whereas a transmembrane sequence is required for membrane protein degradation. Here we characterized these proteins in vitro. Although these mutant enzymes were very low in their activities, they were significantly stimulated by dimethyl sulfoxide, which enabled us to characterize their activities. LacY-FtsH degraded both soluble and membrane proteins, but Zip-FtsH(DeltaTM) only degraded soluble proteins. These proteins also exhibited significant ATPase activities. However, FtsH(DeltaTM) remained inactive both in ATPase and in protease activities even in the presence of dimethyl sulfoxide. The monomeric FtsH(DeltaTM) was able to bind ATP and a denatured protein. These results indicate that subunit association is important for the enzymatic catalysis by FtsH and that the additional presence of the transmembrane sequence is required for this enzyme to degrade a membrane protein even under detergent-solubilized conditions. 相似文献
7.
Van Thong Pham Illimar Altosaar Michael Norman Duhig Harvey Kaplan 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):48-53
Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000. 相似文献
8.
M Ferrer C Woodward G Barany 《International journal of peptide and protein research》1992,40(3-4):194-207
The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges. 相似文献
9.
The delipidated protein moiety, apolipoprotein B, of human low-density lipoproteins was permethylated in potassium butoxide/dimethyl sulfoxide with methyl iodide. The derivatized protein was soluble in dimethyl sulfoxide and, in the presence of sodium dodecyl sulfate, in an aqueous buffer. Analysis of the methylated apolipoprotein B by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate revealed five discrete bands of lower molecular mass than that of the parent 265-kDa protein, which disappeared upon permethylation. The electrophoretic behavior of the methylated apolipoprotein B was distinctly different from that of the other methylated proteins studied, including transferrin, bovine serum albumin, aldolase, beta-lactoglobulin, and apolipoprotein A-I, all of which had a higher apparent molecular weight after permethylation as compared to the corresponding native polypeptide. Calculated on the basis of methylated standard proteins the five polypeptides of apolipoprotein B have apparent molecular masses of 9.0, 16.6, 25.6, 35.7, and 46.7 kDa. The results suggest that the protein moiety of human low-density lipoprotein consists of subunits. In general, the results indicate that the permethylation method can be used to solubilize hydrophobic proteins in organic solvents for structural studies. 相似文献
10.
John H. Northrop 《The Journal of general physiology》1932,16(2):323-337
1. If dilute solutions of purified trypsin of low salt concentration at pH from 1 to 7 are heated to 100°C. for 1 to 5 minutes and then cooled to 20°C. there is no loss of activity or formation of denatured protein. If the hot trypsin solution is added directly to cold salt solution, on the other hand, all the protein precipitates and the supernatant solution is inactive. 2. The per cent of the total protein and activity present in the soluble form decreases from 100 per cent to zero as the temperature is raised from 20°C. to 60°C. and increases again from zero to 100 per cent as the solution is cooled from 60°C. to 20°C. The per cent of the total protein present in the soluble (native) form at any one temperature is nearly the same whether the temperature is reached from above or below. 3. If trypsin solutions at pH 7 are heated for increasing lengths of time at various temperatures and analyzed for total activity and total protein nitrogen after cooling, and for soluble activity and soluble (native) protein nitrogen, it is found that the soluble activity and soluble protein nitrogen decrease more and more rapidly as the temperature is raised, in agreement with the usual effects of temperature on the denaturation of protein. The total protein and total activity, on the other hand, decrease more and more rapidly up to about 70°C. but as the temperature is raised above this there is less rapid change in the total protein or total activity and at 92°C. the solutions are much more stable than at 42°C. 4. Casein and peptone are not digested by trypsin at 100°C. but when this digestion mixture is cooled to 35°C. rapid digestion occurs. A solution of trypsin at 100°C. added to peptone solution at zero degree digests the peptone much less rapidly than it does if the trypsin solution is allowed to cool slowly before adding it to the peptone solution. 5. The precipitate of insoluble protein obtained from adding hot trypsin solutions to cold salt solutions contains the S-S groups in free form as is usual for denatured protein. 6. The results show that there is an equilibrium between native and denatured trypsin protein the extent of which is determined by the temperature. Above 60°C. the protein is in the denatured and inactive form and below 20°C. it is in the native and active form. The equilibrium is attained rapidly. The results also show that the formation of denatured protein is proportional to the loss in activity and that the re-formation of native protein is proportional to the recovery of activity of the enzyme. This is strong evidence for the conclusion that the proteolytic activity of the preparation is a property of the native protein molecule. 相似文献
11.
1. The rate of inactivation of crystalline trypsin solutions and the nature of the products formed during the inactivation at various pH at temperatures below 37°C. have been studied. 2. The inactivation may be reversible or irreversible. Reversible inactivation is accompanied by the formation of reversibly denatured protein. This denatured protein exists in equilibrium with the native active protein and the equilibrium is shifted towards the denatured form by raising the temperature or by increasing the alkalinity. The decrease in the fraction of active enzyme present (due to the formation of this reversibly denatured protein) as the pH is increased from 8.0 to 12.0 accounts for the decrease in the rate of digestion of proteins by trypsin in this range of pH. 3. The loss of activity at high temperatures or in alkaline solutions, just described, is very rapid and is completely reversible for a short time only. If the solutions are allowed to stand the loss in activity becomes gradually irreversible and is accompanied by the appearance of various reaction products the nature of which depends upon the temperature and pH of the solution. 4. On the acid side of pH 2.0 the trypsin protein is changed to an inactive form which is irreversibly denatured by heat. The course of the reaction in this range is monomolecular and its velocity increases as the acidity increases. 5. From pH 2.0 to 9.0 trypsin protein is slowly hydrolyzed. The course of the inactivation in this range of pH is bimolecular and its velocity increases as the alkalinity increases to pH 10.0 and then decreases. As a result of these two reactions there is a point of maximum stability at about pH 2.3. 6. On the alkaline side of pH 13.0 the reaction is similar to that in strong acid solution and consists in the formation of inactive protein. The course of the reaction is monomolecular and the velocity increases with increasing alkalinity. From pH 9.0 to 12.0 some hydrolysis takes place and some inactive protein is formed and the course of the reaction is represented by the sum of a bi- and monomolecular reaction. The rate of hydrolysis decreases as the solution becomes more alkaline than pH 10.0 while the rate of formation of inactive protein increases so that there is a second point at about pH 13.0 at which the rate of inactivation is a minimum. In general the decrease in activity under all these conditions is proportional to the decrease in the concentration of the trypsin protein. Equations have been derived which agree quantitatively with the various inactivation experiments. 相似文献
12.
Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: a simple assay for tryptophan 总被引:1,自引:0,他引:1
Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds. 相似文献
13.
Binding of dimethyl sulfoxide to lysozyme in crystals, studied with neutron diffraction 总被引:1,自引:0,他引:1
Crystals of hen egg white lysozyme soaked in 15% (v/v) dimethyl sulfoxide have been studied with single-crystal neutron diffraction to determine the effect of the solvent molecules on the protein configuration. A total of 9423 statistically significant Bragg reflections to a resolution of approximately 1.8 A were used to locate 6 dimethyl sulfoxide molecules, and structure refinements including a model for the flat solvent lead to a final crystallographic agreement factor of 0.130. The mode of location of the dimethyl sulfoxide molecules was compared with that in previous studies employing ethanol. This showed that hydrophobic interactions can be an essential factor in fixing the probe molecules on the protein surface. There was, however, no sign of any significant change in the protein configuration; so although possibly at higher concentrations of dimethyl sulfoxide the protein will unfold, there was no indication of any precursor effect. 相似文献
14.
Heats of solution for myelin basic protein have been determined using microcalorimetry. All aqueous systems studied yielded negative heats of solution; in contrast, trifluoroethanol produced a small positive heat of solution, while reaction with dimethyl sulfoxide was strikingly exothermic. The heat of interaction for native myelin basic protein with 8 M urea at pH 4.0, 29 degrees C, was found to be -79 +/- 16 kcal/mol. The significance of these results in terms of the protein's structural organization is discussed. 相似文献
15.
Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages 总被引:6,自引:5,他引:1
The isolation of deoxyribonucleic acid (DNA)-binding proteins from various stages of growth and sporulation of Bacillus subtilis is described. After adsorption and elution from phosphocellulose, the proteins were fractionated according to their ability to adsorb to denatured calf thymus DNA-cellulose or native B. subtilis DNA-cellulose. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purification was monitored by a nitrocellulose filter binding assay. Approximately 1% of the proteins in the crude extract adsorbed to denatured calf thymus DNA-cellulose and 0.1% adsorbed to native B. subtilis DNA-cellulose. Each class of proteins varied qualitatively and quantitatively as sporulation proceeded. Several proteins from the exponential phase of growth that bound to denatured DNA were lost by T(0), whereas at T(5) new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and T(0); however, the dominant species in these eluates were clearly different. 相似文献
16.
The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion. 总被引:10,自引:0,他引:10
G E Davis 《Experimental cell research》1992,200(2):242-252
Activated monocytic cells and neutrophils adhere to substrates coated with a wide variety of proteins including albumins, catalase, casein, and various extracellular matrix proteins. This adhesion can be specifically inhibited by antibodies directed to the beta 2 integrin subunit. This adhesion to protein substrates shares some similarities with two known protein-protein recognition systems with little apparent binding specificity, namely, the interactions of heat shock proteins and histocompatibility antigens with denatured proteins or peptides. Cell adhesion and affinity chromatography experiments were performed to test the hypothesis that monocytes and neutrophils adhere to and migrate on protein substrates due to the presence of cell surface receptors that recognize common protein structures such as denatured protein epitopes. Adhesion experiments revealed that activated monocytic cells adhere more rapidly and extensively on substrates coated with denatured protein versus native protein. Both adhesion and migration on such substrates in vitro was dependent on beta 2 integrins since blocking antibodies completely interfered with these cellular responses. Affinity chromatography experiments revealed that the Mac-1 and p150,95 integrins could be isolated from monocyte-differentiated HL-60 cells or neutrophils on a denatured protein-Sepharose column. Much greater yields of the receptors were obtained on a denatured versus native protein Sepharose column. The binding of these receptors was specific in that the LFA-1 beta 2 integrin did not bind to the denatured protein column. These data provide evidence that the adhesion of activated monocytes and neutrophils to many protein substrates in vitro is due to the ability of Mac-1 and p150,95 to directly bind to denatured proteins. A model of leukocyte adhesion and invasion whereby activated leukocytes denature extracellular proteins during diapedesis, making them suitable for recognition by beta 2 integrins, is proposed. 相似文献
17.
Changes in the absorbance spectrum of tetraphenylporphyrin sulfonate (TPPS) are observed that are unique for the proteins lysozyme, luciferase, apomyoglobin, myoglobin, gamma globulin, insulin, RNAase, phosphotransacetylase, papain, ovalbumin, bovine serum albumin (BSA), protamine sulfate, and polylysine. The absorbance spectrum of porphyrins is different for native compared with heat denatured RNAase. A unique absorbance wavelength red shift is observed with trypsin when trypsin inhibitor is present, indicating that porphyrins incorporated with proteins can detect conformational changes in the protein. The absorbance spectrum of the Soret band of TPPS undergoes bathochromic shifts upon addition of local anesthetics to acetylcholine esterase (AChE), suggesting that the absorbance spectrum of porphyrins can be used as a reporter of the presence of inhibitors of AChE by indicating conformational changes on binding of the inhibitor. 相似文献
18.
Effects of aphidicolin on retrovirus DNA synthesis in vivo 总被引:4,自引:0,他引:4
K Hagino-Yamagishi K Kano Y Mano 《Biochemical and biophysical research communications》1981,102(4):1372-1378
Renaturation of green-fluorescent protein (A-GFP) was achieved for the first time following denaturation in guanidine-HCl or acid. Denaturation was accompanied by the concerted loss of visible fluorescence, alteration of absorption characteristics, and large negative deflection of CD signal in the far UV. Dialysis of a guanidine-denatured sample at pH 8 resulted in 64% renaturation (return to native absorption) and neutralization of an acid-denatured sample restored 90% of the native absorption. Renatured GFP is highly fluorescent and indistinguishable from native GFP with respect to the shape of excitation and emission spectra. Both native and denatured proteins exhibit resistance to trypsin hydrolysis and have identically broad pH and heat stability profiles, all of which suggest full renaturation. 相似文献
19.
All of the nine cysteine residues in dimethyl sulfoxide reductase(OMSOR) exist in reduced thiol form. The unfolded form, whichwas previously detected in DMSOR proteins secreted by spheroplastsprepared from a molybdenum cofactor-deficient mutant, was alsodetected in spheroplasts from a wild type strain when iodoacetamidewas present, suggesting that DMSOR is secreted first in a reducedand unfolded form. In spheroplasts from the mutant, a new foldingintermediate migrating between the unfolded and native formswas additionally detected on non-denaturing gel. This intermediatecontained no disulfide bonds, but had a folded compact conformationsimilar to that of the native form. (Received October 4, 1996; Accepted September 4, 1997) 相似文献
20.
Blue native gel electrophoresis (BN–PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN–PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN–PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. 相似文献