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1.
The α/β Interfaces of α1β1, α3β3, and F1: Domain Motions and Elastic Energy Stored during γ Rotation
ATP synthase (FoF1) consists of F1 (ATP-driven motor) and Fo (H+-driven motor). F1 is a complex of 33 subunits, and is the rotating cam in 33. Thermophilic F1 (TF1) is exceptional in that it can be crystallized as a monomer and an 33 oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F1 (MF1) and TF1 were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF1) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in FoF1 has been revised. X-ray crystallography of F1 is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of FoF1. The domain motion and elastic energy in FoF1 will be elucidated by time-resolved crystallography. 相似文献
2.
A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of 18O2 into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC
thin layer-chromatography
- PIPES
piperazine-N,N-bis-(2-ethanesulfonate) Na
- TES
2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid
Dedicated to Professor G. Drews on occasion of his 60th birthday 相似文献
3.
Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of 13C and 13C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of 13C and 13C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of 13C and 13C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides. 相似文献
4.
D. F. Stiffler S. Eskandari 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(5):355-361
Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO3
-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO2 during the acidosis. The 1 and 2 antagonists, timolol and butoxamine respectively, (0.2 g·g-1) were administered to separate groups of larvae. Butoxamine (2) delayed the recovery from the acidosis by prolonging the increase in arterial PCO2 and reversing the recovery of [HCO3
-]. Timolol (1) did not delay recovery. We conclude that 2 receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC
red blood cell 相似文献
5.
Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and L-crystallins was studied during thermal denaturation of L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of L-crystallin it binds with modified -crystallin or, alternatively, L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -L-crystallin complex size and relative contents of - and -L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation. 相似文献
6.
Hans-Peter E. Kohler 《Archives of microbiology》1988,150(3):219-223
Methanothrix soehngenii was found to contain five different cobamides when grown on vitamin B12 supplemented as well as vitamin B12 free media. In both cases, it was shown by HPLC-chromatography and UV/VIS spectroscopy, that -5-methylbenzimidazolyl--cyanocobamide was the predominant cobamide, accounting for 27% and 23%, respectively, of the total corrinoid content. Vitamin B12 and -5-hydroxybenzimidazolyl--cyanocobamide could also be detected in both cell batches in varying amounts. Cells grown on vitamin B12 free medium contained significantly more baseless cobamides, indicating biosynthesis of cobamides.Abbreviations (5-MeBza)CNCba
-5-methylbenzimidazolyl--cyanocobamide
- (5-HOBza)CNCBa
-5-hydroxybenzimidazolyl--cyanocobamide (factor III)
- (5-MeOBza)CNCba
-5-methoxybenzimidazolyl--cyanocobamide (factor IIIm)
- (Bza)CNCba
-benzimidazolyl--cyanocobamide 相似文献
7.
-Carotene synthesis was increased from a negligible amount to 152 mg (g-dry cells)–1 and H2O2 was accumulated up to 16.7 M during 2.5 day-culture of Blakeslea trispora. When cells were cultivated in 250 ml flasks containing various volumes (25–150 ml) of the medium, not only H2O2 accumulation but also -carotene synthesis increased as culture volume decreased. Addition of H2O2 (10 M) to the 1.5-day old cultures of B. trispora resulted in 46% higher -carotene synthesis than that without addition. All these results indicate that -carotene biosynthesis is stimulated by H2O2 in B. trispora. 相似文献
8.
The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl2, -1-3-linked glucans are mainly produced. By increasing MgCl2 concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl2, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl2.Abbreviations G.S. I
glucan synthetase I
- G.S. II
glucan synthetase II
- Dol. P
dolichol phosphate 相似文献
9.
Lin CC Sun CC Luo SF Tsai AC Chien CS Hsiao LD Lee CW Hsieh JT Yang CM 《Journal of biomedical science》2004,11(3):377-390
Interleukin-1 (IL-1) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1-induced cyclooxygenase (COX) expression and PGE2 synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1 increased COX-2 expression and PGE2 synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1-induced responses in HTSMCs. IL-1-induced COX-2 expression and PGE2 synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1. Furthermore, IL-1-induced activation of nuclear factor-B (NF-B) was inversely correlated with the degradation of IB- in HTSMCs. IL-1-induced COX-2 expression and PGE2 synthesis were inhibited by the NF-B inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE2 from IL-1-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-B signaling pathways in HTSMCs. 相似文献
10.
-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor. 相似文献
11.
ATP synthase (F0F1) is driven by an electrochemical potential of H+ (H+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is 33. The active 33 oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F0. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of 33. The torque of the rotation is estimated to be 420 pN·å from the H+ and H+-current through F0F1. The angular velocity () of is the rate-limiting step, because H+ increased theV
max of H+ current through F0, but not theK
m
(ATP). The rotational unit of F0 (=ab2c10) is /5, while that in 33 is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN3-ATP), and tight Mg-ATP binding in 33 was shown by ESR. The rotational relaxation of TF1 is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of 33 measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF1) and F0-d to hold free rotation of 33. 相似文献
12.
The Ca2+ channel 1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, 1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel 1 or subunit. In this study, we studied the levels of 1A, 1C, 1D, 1E, 1, 2, 3 and 4 mRNAs in adrenal gland of 1B-deficient mice. The 1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of 1C, 1D, 1E, 1, 2, 3 and 4 was found among wild, heterozygous and homozygous mice. The protein level of 1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of 1A gene, we examined lacZ expression in 1B-deficient × 1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of 1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous 1B-deficient × 1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of 1B-deficient mice is that compensation by 1A subunit occurs and that 6.3-kb 5-upstream region of 1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005) 相似文献
13.
Cowburn Richard F. Wiehager Birgitta Trief Ewa Li-Li Mo Sundström Erik 《Neurochemical research》1997,22(12):1437-1442
The neurotoxic fragment corresponding to residues 25-35 of the -amyloid (A) peptide [A-(25-35)] has been shown to exert effects on (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([3H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A-(25-35) [A-(25-35-NH2) and A-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A-(25-35-NH2) and A-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [3H]glutamate and [3H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten M A-(25-35-NH2) and A-(25-35-COOH) gave 25% and 20% inhibitions of [3H]glutamate binding and 75% and 70% inhibitions of [3H]glycine binding, respectively. A-(25-35-NH2), but not A-(25-35-COOH), gave a small (ca. 17% at 10 M) statistically significant increase of [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding. [3H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC50 value for calcium stimulation of [3H]nitrendipine binding. It is concluded that A-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels. 相似文献
14.
To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t
1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t
1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l–1 over the same period. 相似文献
15.
2-Macroglobulin (2M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the 2M sequence. In the 3D-structure of 2M, TGF- occupies the 2M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary 2M–protease complexes (2 mol protease/mol 2M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to 2M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary 2M–trypsin complex (1 mol trypsin/mol 2M) binds increased amounts of TGF-1, compared with native 2M, while ternary 2M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary 2M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native 2M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the 2M central cavity; however, 2M–plasmin complex also bound increased amounts of A, compared with native 2M. We conclude that A accesses its binding site, in 2M, from outside the 2M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of 2M, but also in the 3D-structure. 相似文献
16.
Kaumann Alberto J. Sanders Louise Lynham James A. Bartel Sabine Kuschel Meike Karczewski Peter Krause Ernst-Georg 《Molecular and cellular biochemistry》1996,163(1):113-123
Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both 1-adrenoceptors (1AR) and 2-adrenoceptors (2AR) mediate positive inotropic effects but that only 1AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both 1 AR and 2 AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of 1AR and 1/3 of 2AR, whether or not 2AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate 2AR, we used the 2AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t1:2 of relaxation) effects with EC50 values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the 2AR-selective antagonist ICI 118551 (50 nM) which reduced both EC50 values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC50 of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[125I] cyanopindolol with higher affinity for 2AR than for - 1 AR; the binding to 2AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both 1AR and 2AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both 1AR and 2AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation. 相似文献
17.
The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family. 相似文献
18.
The gene encoding -mannanase was cloned from alkalophilic Bacillus sp. AM-001 into Escherichia coli JM 101 by inserting HindIII-generated DNA fragments into the HindIII site of pUC19. A 2.0 kb XbaI-PstI fragment of the donor strain DNA was sufficient for -mannanase synthesis. The amount of -mannanase expressed in E. coli JM101 harboring pMAH3 (containing a 2.4 kb XbaI-HindIII fragment) was about 24% of the activity produced by the donor strain. E. coli JM101 harboring pMAH3 was found to produce two enzymatically active -mannanases (A and B). These two -mannanases were purified to electrophoretically homogenous states. The -mannanase A had enzymatic properties similar to those of the -mannanases M-I and M-II produced by alkalophilic Bacillus sp. AM-001, and the -mannanase B resembled its -mannanase M-III. In contrast to -mannanase production in the donor strain, that in E. coli was not inducible. The NH2-terminal amino acid sequences from amino acid 1 (Asn) to 9 (Gln) of the three -mannanases purified from alkalophilic Bacillus sp. AM-001 coincide with those from amino acid 4 (Asn) to 12 (Gln) of the two -mannanases purified from E. coli transformant. 相似文献
19.
Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at 11 contact is 55 MetSer. 相似文献
20.
Pasquale Ferranti Andrea Scaloni Simonetta Caira Lina Chianese Antonio Malorni Francesco Addeo 《Journal of Protein Chemistry》1998,17(8):835-844
The primary structure of water buffalo s1-casein and of -casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a -elimination/thiol derivatization. Water buffalo s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine s1-casein C variant, the water buffalo s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine A2-casein variant, the two water buffalo -casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo -casein variants seem to be homologous to bovine A2-casein. 相似文献