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1.
The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation. 相似文献
2.
Deglycosylation was shown to be an important prerequisite step for the crystallization of glucose oxidase from Aspergillus niger. Whereas the glycosylated enzyme could not be crystallized, crystals of the deglycosylated enzyme suitable for X-ray diffraction analysis were reproducibly grown in the presence of 1.6 M-ammonium sulphate and octanetriol at pH 5.3 to 5.6. The crystals belong to the space group P3(1)21 or P3(2)21 with refined lattice constants of a = 66.5 A and c = 214.4 A, indicating a cell content of one monomer per asymmetric unit of the crystal. Crystals diffract to at least 2.5 A resolution. Cleavage of 95% of its carbohydrate moiety affected the kinetics of glucose oxidation, stability at low pH and some electrophoretic properties of glucose oxidase, such as molecular mass and the number of isoelectric forms. However, other properties, such as thermal stability, pH and temperature optima of activity were not affected. 相似文献
3.
J Hendle H J Hecht H M Kalisz R D Schmid D Schomburg 《Journal of molecular biology》1992,223(4):1167-1169
The dimeric glucose oxidase from Penicillium amagasakiense was deglycosylated, purified and crystallized as a complex with its coenzyme FAD. Deglycosylation and purification to isoelectric homogeneity were shown to be an important prerequisite step to obtain crystals suitable for X-ray investigations. Crystals of the deglycosylated enzyme were reproducibly grown using ammonium sulfate as precipitant at pH 7.4 to 7.5. Crystals diffract to at least 2.0 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with refined lattice constants of a = 59.3 A, b = 136.3 A and c = 156.7 A. Assuming two monomers (approximately 135 kDa) per asymmetric unit the Vm value is 2.3 A3/Da. 相似文献
4.
K Shinzawa-Itoh H Yamashita S Yoshikawa Y Fukumoto T Abe T Tsukihara 《Journal of molecular biology》1992,228(3):987-990
Fully reduced and CO-bound fully reduced forms of cytochrome c oxidase from beef heart muscle were crystallized in the presence of sodium ascorbate under N2 or CO atmosphere. Hexagonal bipyramidal and tetragonal crystals were obtained for both forms depending on buffer species. The hexagonal bipyramidal crystals, as large as 0.6 mm in the largest dimension, diffracted X-rays at 7 A resolution, showing an identical space group and cell dimension, P6(2) or P6(4) and a = b = 209 A, c = 283 A, respectively. These parameters coincide with those for crystals of the fully oxidized resting enzyme. This result suggests that a large conformational change, like a subunit arrangement, is not induced by the redox change and/or binding of CO (and possibly O2) to heme a3. 相似文献
5.
Fui Chin Chong Beng Ti Tey Zanariah Mohd Dom Kok Hing Cheong Budiatman Satiawihardja Mohd Nordin Ibrahim Russly Abdul Rahman Dayang Radiah Awang Biak Tau Chuan Ling 《Biotechnology and Bioprocess Engineering》2007,12(3):250-256
Rice bran lipase (RBL) was delipidated to enhance its stability in organic solvent and its esterification activity at elevated
temperature. The esterification activity of delipidated RBL increased as temperature was increased from 45 to 65°C. The esterification
activity of delipidated RBL at 65°C was about 14 times greater than that of the non-delipidated RBL. As temperature was further
increased to 75°C, the non-delipidated RBL lost all esterification activity, whereas the delipidated RBL retained approximately
48% of its esterilication activity. The delipidated RBL maintained a relative esterification activity greater than 80% after
16 h of incubation in hexane, whereas the non-delipidated RBL maintained a relative esterification activity of only 50%. A
method for production of acylglycerol using delipidated RBL to esterify palm oil fatty acid distillate (PFAD) with glycerol
in hexane was successfully developed. The effects of reaction temperatures and type of water removal agents (silica gel and
molecular sieve) on the degree of esterification were also examined. A 4 h reaction at 65°C, catalyzed by delipidated RBL
and using silica gel as the water removal agent resulted in 53.8% esterification. Thin layer chromatography analysis suggested
that the esterified product was primarily comprised of mono-and di-acylglycerols. 相似文献
6.
Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl.
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Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin. 相似文献
7.
The two-chain form of human cathepsin D was purified from human spleen with a method utilizing an ion exchange chromatography step prior to the pepstatin affinity column normally used to purify aspartic proteases. The protein was crystallized from 21% polyethylene glycol 8000 at pH 4.0 using the hanging drop vapour diffusion method. Small crystals were used as seeds to grow crystals suitable for X-ray data collection. The crystals diffract to a resolution of 3.2 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 99.6 A, c = 133.6 A. There are two molecules in the asymmetric unit. 相似文献
8.
The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc. 相似文献
9.
H Sobek T Haag O Pfeifer D Schomburg F Lingens K H van Pée 《Journal of molecular biology》1991,221(1):35-37
Bromoperoxidase from Streptomyces aureofaciens ATCC 10762, a non-haem haloperoxidase, has been crystallized using the hanging drop method. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group P2(1)3 with a = 123.4 A. The asymmetric unit contains a dimer of Mr = 60,200. The crystals diffract to at least 2.3 A resolution and are suitable for crystallographic structure analysis. 相似文献
10.
The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 Å, b = 122.3 Å, c = 68.0 Å, and there is one ncd molecule per asymmetric unit. The crystals diffract X-ray to at least 2.3 Å and are appropriate for high-resolution structure determination. © 1995 Wiley-Liss, Inc. 相似文献
11.
12.
Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit. 相似文献
13.
Ewa Tykarska Lukasz Lebioda Elzbieta Marchut Janusz Steczko Boguslaw Stec 《The protein journal》1990,9(1):83-86
Alcohol oxidase fromPichia pastoris has been crystallized from polyethylene glycol 4000 solutions. The crystals are tetragonal, a=228 Å, c=456 Å space groupP41212. The crystals scatter only to about 6 Å resolution; their poor crystallinity may have some physiological function. Secondary structure predictions suggest that the C-terminal part of the molecule, residues 311–664, has the folding of an eightfold β/α-barrel (TIM barrel). This would indicate common ancestry with four other flavoenzymes: canavalin, glycolate oxidase, flavocytochrome b, and trimethylamine dehydrogenase. 相似文献
14.
Crystallization and preliminary X-ray analysis of Gc, the vitamin D-binding protein in serum 总被引:1,自引:0,他引:1
The vitamin D-binding protein, Gc, was purified from human serum and crystallized using the hanging-drop method. The best crystals were grown from 28% polyethylene glycol 400 in 50 mM-sodium acetate at pH 4.8. These crystals diffract to 3.4 A and the observed diffraction is consistent with orthorhombic space groups P4(1) and P4(3). The unit cell parameters were determined to be a = b = 135.5 A and c = 75.6 A. 相似文献
15.
Crystallization and preliminary X-ray study of subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex 总被引:1,自引:0,他引:1
Subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex was dissociated from the complex, purified and crystallized using the hanging- or sitting-drop method of vapour diffusion, with ammonium sulphate as the precipitant. The assays were carried out at pH 4.2 (20 mM-acetate buffer). An X-ray examination of the crystals shows that they are monoclinic, with a space group P21 and cell dimensions a = 47.9 A, b = 61.3 A, c = 39.0 A and beta = 95.0 degrees. The asymmetric unit contains one molecule of 25,800 Mr. The crystals are suitable for structure determination to at least 2.8 A resolution. 相似文献
16.
A Rodríguez M Tablero B Arreguín A Hernández R Arreguín M Soriano-García A Tulinsky C H Park T P Seshadri 《The Journal of biological chemistry》1988,263(9):4047-4048
Hevein, a small protein from the latex of Hevea brasiliensis, has been crystallized by the vapor diffusion method using 2-methyl-2,4-pentanediol and CaCl2 as the precipitant agents. The crystals are orthorhombic space group P21212 with a = 21.88, b = 31.90, and c = 51.24 A and one molecule in the asymmetric unit. The crystals are quite stable to x-rays and suitable for a high resolution three-dimensional structure determination. 相似文献
17.
The human chloride intracellular channel protein CLIC4 has been crystallized by the hanging-drop vapour-diffusion technique using trisodium citrate as the precipitant. The best crystals were obtained by the microseeding method. The crystals diffracted to 2.2 A resolution and were found to belong to space group P121, with unit-cell parameters a = 73.19, b =86.05, c = 73.38 A, beta = 112.99 degrees and three molecule per asymmetric unit. 相似文献
18.
The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of beta-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 degrees C. The orthorhombic crystals (space group P2(1)2(1)2(1)) have cell constants a = 142.5 A, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region. 相似文献
19.
Carolyn Ozment Joseph Barchue Lawrence J. DeLucas Debasish Chattopadhyay 《Journal of structural biology》1999,127(3):279-282
Escherichia coli NAD synthetase was overexpressed and purified to homogeneity. The recombinant protein was active in an in vitro enzyme assay. The enzyme required approximately 1.5 mM magnesium for optimal activity. The pH optimum was found to be 8.0-8.5. The recombinant protein was crystallized at room temperature using the hanging-drop vapor diffusion technique with 1.5 M lithium sulfate, 0. 1 M Hepes buffer at pH 7.5 as precipitant. The protein was also crystallized in the presence of its substrates, nicotinic acid adenine dinucleotide and adenosine triphosphate under similar conditions. These crystals diffract to 2.0-A resolution and belong to trigonal space group P3(1)21 with unit cell dimensions of a = b = 91.766, c = 74.17 A and alpha = beta = 90 degrees, gamma = 120 degrees. The structure of the complex has been determined using the molecular replacement method. 相似文献
20.
Crystallization and a preliminary X-ray crystallographic study of alpha-amylase from Bacillus licheniformis 总被引:1,自引:0,他引:1
alpha-Amylase from Bacillus licheniformis has been crystallized by the hanging-drop vapor diffusion method in the presence of calcium ions using ammonium sulfate as precipitant. The crystals are tetragonal, belonging to the space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions of a = 119.9 and c = 85.4 A. The asymmetric unit contains one molecule of alpha-amylase, with a crystal volume per protein mass (VM) of 2.78 A3/Da. The crystals diffract to better than 2.0 A Bragg spacing when exposed to synchrotron X-rays and they are reasonably stable in the X-ray beam. Thus the crystals are suitable for structure determination at high resolution by X-ray methods. 相似文献