共查询到20条相似文献,搜索用时 15 毫秒
1.
Bernard N. Violand Mark Takano Daniel F. Curran Larry A. Bentle 《Journal of Protein Chemistry》1989,8(5):619-628
A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein. 相似文献
2.
Formation of isoaspartate 99 in bovine and porcine somatotropins 总被引:4,自引:0,他引:4
Bernard N. Violand Michael R. Schlittler Paul C. Toren Ned R. Siegel 《Journal of Protein Chemistry》1990,9(1):109-117
Asparagine 99 in bovine (BST) and porcine somatotropins (PST) was converted to an isoaspartate residue during incubation at neutral or alkalinepH. Isoaspartate 99 BST or isoaspartate 99 PST was resolved from the normal somatotropin by reversed-phase high-performance liquid chromatography (HPLC). The altered peptide of residues 96–108 which contains isoaspartate 99 was detected by tryptic peptide mapping of the modified BST or PST. Amino acid sequencing, amino acid analysis, mass spectrometry, and co-elution with a chemically synthesized peptide containing isoaspartate 99 were used to demonstrate the existence of isoaspartate in the modified peptides. Peptide bond cleavage between Asn 99 and Ser 100 also occurred during incubation of BST and PST at neutral or alkalinepH. This chemically cleaved product was resolved on reversed-phase HPLC from both the isoaspartate 99 and normal somatotropin molecules. 相似文献
3.
A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways. 总被引:1,自引:1,他引:1
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J. A. Wilkins J. Cone Z. I. Randhawa D. Wood M. K. Warren H. E. Witkowska 《Protein science : a publication of the Protein Society》1993,2(2):244-254
The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates. 相似文献
4.
Gregg Bogosian Patricia J. L. Morris Michael D. Hale James F. Kane 《Journal of industrial microbiology & biotechnology》1992,9(1):27-36
Summary The fate in water ofEscherichia coli K-12 strain LBB269, both plasmid-free and carrying the recombinant plasmid pBGH1, was studied.E. coli K-12 strain LBB269 (pBGH1) is a nalidixic acid resistant derivative of W3110G (pBGH1), the microorganism used by Monsanto Company for the commercial production of bovine somatotropin. Water samples were obtained from the Missouri River and from the Monsanto Life Sciences Research Center aqueous waste basin. Strains LBB269 and LBB269 (pBGH1) were grown in fermentation vessel under bovine somatotropin (BST) production conditions, and inoculated into the water samples. The inoculated water samples were incubated, at 26°C, and the number of viableE. coli cells was determined as a function of time. In sterile water from both sources, the two strains remained, at a constant level for at least 28 days; LBB269 (pBGH1) remained at a constant level in sterile water for at least 300 days. In non-sterile water from both sources, the two strains declined from an initial concentration of about 3.0×106 cells per ml to less than 10 cells per ml in 147 h. The study conditions did not adversely affect the populations of indigenous microorganisms. The selective loss of strains LBB269 and LBB269 (pBGH1) demonstrates that theseE. coli strains do not survive in environmental sources of water. In addition, it was observed that the presence of pBGH1 had essentially no effect on the disappearance of strain LBB269 from either source of water. 相似文献
5.
Michael A. Heitkamp James F. Kane Patricia J. L. Morris Martina Bianchini Michael D. Hale Gregg Bogosian 《Journal of industrial microbiology & biotechnology》1993,11(4):243-252
Summary The fate of a derivative ofEscherichia coli strain W3110G [pBGH1], a strain used for production of bovine somatotropin, was examined in semi-continuous activated sludge (SCAS) units. A nalidixic acid-resistant derivative of W3110G [pBGH1], strain LBB270 [pBGH1], was used to facilitate tracking. SCAS units (300 ml) containing municipal mixed liquor were operated on a daily cycle of 23 h aeration and 1 h settling followed by decanting of clear supernatant (175 ml) and refilling with fresh primary effluent. SCAS units were inoculated with two concentrations ofE. coli LBB270 [pBGH1] and operated for 200 h. Viable levels ofE. coli LBB270 [pBGH1] were measured daily in aerated mixed liquor and decanted supernatant. Viable counts in the mixed liquor decreased from 10000- to 100000-fold in less than 200 h. Losses ofE. coli LBB270 [pBGH1] in decanted supernatants accounted for less than 2-fold of the total losses observed in the SCAS units. TheE. coli LBB270 [pBGH1] was not evenly distributed in the mixed liquor, but became preferentially associated with the settleable floc. These results show thatE. coli LBB270 [pBGH1] was unable to survive in municipal sludge even when inoculated at concentrations greater than, or comparable to, levels of indigenous microorganisms. 相似文献
6.
Sá Filho MF Carvalho NA Gimenes LU Torres-Júnior JR Nasser LF Tonhati H Garcia JM Gasparrini B Zicarelli L Baruselli PS 《Animal reproduction science》2009,113(1-4):51-59
The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs. 相似文献
7.
Although much work has been reported on modification of human somatotropin with plasmin and has revealed important information about structure-function relationships, plasmin modification of nonprimate somatotropins (which differ markedly in structure and biological actions from the human hormone) has been little studied. Therefore we investigated plasmin digestion of bovine somatotropin. Digestion was less rapid but more extensive than that of human somatotropin. Structural characterization of digestion products resolved by gel filtration suggested that a major product after 24 h was a disulfide-linked fragment comprising residues 1–133 plus 140–177. Further cleavage products were found in aggregated material and minor fractions. In radioimmunoassay for bovine somatotropin, activity was retained only by the unfractionated digest and aggregated material. In radioreceptor assay for somatotropin using receptors from livers of late-pregnant rabbit all preparations retained some activity. The results suggest that receptor- and antibody-binding sites in bovine somatotropin are not identical and that greater activity may result from specific association of fragments that are less active or inactive once separated. 相似文献
8.
Breaking symmetry in the structure determination of (large) symmetric protein dimers 总被引:2,自引:2,他引:0
We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers. 相似文献
9.
Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129. 总被引:3,自引:3,他引:0
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B. N. Violand M. R. Schlittler E. W. Kolodziej P. C. Toren M. A. Cabonce N. R. Siegel K. L. Duffin J. F. Zobel C. E. Smith J. S. Tou 《Protein science : a publication of the Protein Society》1992,1(12):1634-1641
Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides. 相似文献
10.
Bovine somatotropin (bST) was secreted from Escherichia coli at moderate levels of 1-2 micrograms/ml/OD using expression vectors in which the bST gene was fused to the lamB secretion signal. To study the secretion properties of bST in E.coli further, two approaches for modifying the secretion signal were employed. In the first case, fusion proteins were constructed with six alternative bacterial secretion signals: three from E.coli proteins (HisJ, MalE and OmpA), two from bacteriophage proteins (M13 coat protein and PA-2 Lc) and one from the chitinase A protein of Serratia marcescens. The results, as monitored by Western blot analysis of both total cell protein and the periplasmic fraction, showed that these changes in the secretion signal did not significantly affect the secretion properties of bST. In the second approach, a library of random mutations was created in the lamB secretion signal and 200 independent clones were screened. The level of secreted bST was determined by growing individual clones in duplicate in microtiter wells, inducing protein expression and measuring the bST released by osmotic shock using a particle concentration fluorescent immunoassay. The secretion properties of several novel variants in the LamB signal peptide are presented. 相似文献
11.
B. A. Becker H. D. Johnson R. Li R. J. Collier 《International journal of biometeorology》1990,34(3):151-156
A study was conducted to evaluate the effect of bovine somatotropin (BST) supplementation in twelve lactating dairy cows maintained in cold environmental conditions. Six cows were injected daily with 25 mg of BST; the other six were injected with a control vehicle. Cows were maintained under standard dairy management during mid-winter for 30 days. Milk production was recorded twice daily, and blood samples were taken weekly. Animals were then transferred to environmentally controlled chambers and exposed to cycling thermoneutral (15° to 20° C) and cycling cold (–5° to +5° C) temperatures for 10 days in a split-reversal design. Milk production, feed and water intake, body weights and rectal temperatures were monitored. Blood samples were taken on days 1, 3, 5, 8 and 10 of each period and analyzed for plasma triiodothyronine (T3), thyroxine (T4), cortisol, insulin and prolactin. Under farm conditions, BST-treated cows produced 11% more milk than control-treated cows and in environmentally controlled chambers produced 17.4% more milk. No differences due to BST in feed or water intake, body weights or rectal temperatures were found under laboratory conditions. Plasma T3 and insulin increased due to BST treatment while no effect was found on cortisol, prolactin or T4. The results showed that the benefits of BST supplementation in lactating dairy cows were achieved under cold environmental conditions. 相似文献
12.
Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.
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Z. Yu G. Friso J. J. Miranda M. J. Patel T. Lo-Tseng E. G. Moore A. L. Burlingame 《Protein science : a publication of the Protein Society》1997,6(12):2568-2577
Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb. 相似文献
13.
Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A
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Xiong Y Juminaga D Swapna GV Wedemeyer WJ Scheraga HA Montelione GT 《Protein science : a publication of the Protein Society》2000,9(2):421-426
Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A. 相似文献
14.
Friederike Plath Philippe Ringler Alexandra Graff-Meyer Henning Stahlberg Matthias E. Lauer Arne C. Rufer 《MABS-AUSTIN》2016,8(5):928-940
The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs. 相似文献
15.
Müller JJ Hannemann F Schiffler B Ewen KM Kappl R Heinemann U Bernhardt R 《Journal of inorganic biochemistry》2011,105(7):957-965
The protein Etp1 of Schizosaccharomyces pombe consists of an amino-terminal COX15-like domain and a carboxy-terminal ferredoxin-like domain, Etp1fd, which is cleaved off after mitochondrial import. The physiological function of Etp1fd is supposed to lie in the participation in the assembly of iron-sulfur clusters and the synthesis of heme A. In addition, the protein was shown to be the first microbial ferredoxin being able to support electron transfer in mitochondrial steroid hydroxylating cytochrome P450 systems in vivo and in vitro, replacing thereby the native redox partner, adrenodoxin. Despite a sequence similarity of 39% and the fact that fission yeast is a mesophilic organism, thermodynamic studies revealed that Etp1fd has a melting temperature more than 20 °C higher than adrenodoxin. The three-dimensional structure of Etp1fd has been determined by crystallography. To the best of our knowledge it represents the first three-dimensional structure of a yeast ferredoxin. The structure-based sequence alignment of Etp1fd with adrenodoxin yields a rational explanation for their observed mutual exchangeability in the cytochrome P450 system. Analysis of the electron exchange with the S. pombe redox partner Arh1 revealed differences between Etp1fd and adrenodoxin, which might be linked to their different physiological functions in the mitochondria of mammals and yeast. 相似文献
16.
John O. Hui John Le Viswanatham Katta Robert Rosenfeld Michael F. Rohde Mitsuru Haniu 《Journal of Protein Chemistry》1996,15(4):351-358
Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors. 相似文献
17.
Kayoko Fukuda Ikuo Kawashima Motowo Tomita Akira Hamada 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(2):278-288
The O-glycosidically-linked carbohydrate units of glycophorin from bovine erythrocyte membrane were released by alkaline borohydride treatment. These oligosaccharides were separated into the neutral fractions and the acidic fractions by ion-exchange chromatography followed by gel filtration. The two acidic fractions (fractions 10 and 13) which have the smallest molecular weight in acidic oligosaccharides, were further purified by gel filtration on Bio-Gel P-4 column. Two acidic oligosaccharides (fractions 10-I and 10-II), heptasaccharides, were separated by gel filtration on a Bio-Gel P-4 column from fraction 10. These structures were determined by methylation analyses, nitrous acid deamination after hydrazinolysis and Smith degradation after desialylation. In addition, the structures were also analyzed by direct-probe mass spectrometry of the permethylated derivatives before and after desialylation. These studies indicated that one of them (fraction 10-I) was NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) GalNAcol and another heptasaccharide (fraction 10-II) was Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3) [NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→6)]GalNAcol. Athough another acidic fraction (fraction 13) was obtained as a single peak on a Bio-Gel P-4 column, it appeared to be the mixture of a heptasaccharide, NeuNGcα(2→3)Galβ(1→4)GlcNAcβ(1→3 or 6)[Galβ(1→4)GlcNAcβ(1→6 or 3)]Galβ(1→3)GalNAcol and an oligosaccharide similar to fraction 10-II, by analysis of two products obtained by Smith degradation after desialylation. 相似文献
18.
Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1H-15N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured alpha-helical sub-domain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands. 相似文献
19.
Yokota A Takenaka H Oh T Noda Y Segawa S 《Protein science : a publication of the Protein Society》1998,7(8):1717-1727
Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c. 相似文献
20.
A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose
followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide
gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels
resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis
on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence
was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme.
On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary
data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the
[125I ]-labelled protein. The purified enzyme was a glycoprotein and had aK
m
of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme
activity. Mn2+ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration 相似文献