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1.
The solid-state conformation of the first N-protected ornithine derivative has been established by X-ray analysis. The hydrochloride of N-(9-fluorenyl)methoxycarbonyl-L-ornithine crystallises as diethyl ether solvate. The backbone (0, , , 1) torsion angles are (174.9°, –84.0°, 145.9°, –171.0°). The conformation of the urethane amide bond is trans. The ornithine aliphatic side chain adopts preferred fully extended conformation which is stabilised by the hydrogen bonding of the –NH3 + group to the diethyl ether molecule, carboxyl group and Cl- anions.  相似文献   

2.
Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B1 (FB1) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB1 proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.  相似文献   

3.
In natural ecosystems, differences often exist in the relative abundanceof stable S isotopes (°34S) that can provide clues as tothe source, nature, and cycling of S. Values of °34S inprecipitation, throughfall, soils, soil solution, and stream waters weremeasured at the Hubbard Brook Experimental Forest (HBEF), New Hampshire.Values of °34S in precipitation and throughfall weresimilar to each other but differed seasonally. Precipitation°34S values were higher in the dormant season[°34S = 5.9±0.6 (17)][Mean + SE(N)]than in the growing season [°34S = 5.0±0.6(40)] but throughfall growing-season values were higher[°34S = 5.6±0.6(68)] than for the dormantseason [°34S = 4.9±0.7 (9)]. Different treespecies did not affect throughfall °34S values. In soilsolution, °34S values were higher in the growing season(°34S = 8.9±2.8; 8.8±1.7;and 4.0±0.6 for Oa, Bh, and Bs horizons, respectively) thanin the dormant season (°34S = 5.6±1.5;3.7±2.4; and 3.4±1.2 for Oa, Bh, and Bshorizons, respectively). These seasonal differences in°34S were probably caused by biological isotopicfractionation. The °34S values in streams were generally2 lower and more variable than those in precipitation andthroughfall, suggesting fractionation and/or different isotopic sources inthe soil.  相似文献   

4.
1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the -CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3-and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the -CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation. The only changes observed are the -CH2 proton resonances of the metal-coordinating cysteines which are sensitive to local conformational changes within the ternary complex Co(II)-HLADH · Imidazole · NADH in the open conformation or global changes in the ternary complex Co(II)-HLADH · Pyrazole · NAD+ in the closed conformation. Acetate which can be regarded as a substrate model was shown to induce a similar change in the optical spectra of the Co(II) enzyme as all other anions observed so far. From the optical changes a dissociation constant of acetate at the catalytic metal site of 200±50 mM was calculated and from the changes of the 13C-NMR linewidth of 13C acetate direct bonding of the anion to the catalytic Co(II) ion can be demonstrated to occur under the conditions of rapid exchange. The implications of these data for the assessment of tetracoordination around the catalytic metal ion as well as the chemical nature of intermediates occurring along the catalytic pathway are discussed.This work has been performed with contribution of the project Projetto Strategico Biotechnologie CNR and with financial support from the Deutsche Forschungsgemeinschaft, NATO, Bundesminister für Forschung und Technologie, and the Universität des Saarlandes  相似文献   

5.
Summary The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as 3JH n H coupling constants, amide exchange and 13C and 13C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.  相似文献   

6.
Summary The effects of catecholamines on the vascular resistance of the gills, the gas gland and the tail of the Atlantic cod, were studied in isolated preparations perfused at a constant pressure. Adrenaline caused a biphasic response of the gill vasculature with an initial constriction followed by a dilation. The noradrenaline response was usually dilatory, but constrictory responses at all or some concentrations were occasionally seen in some specimens. The branchial dilation was caused by adrenaline concentrations even lower than those found in normal cod plasma (30 nM). The gas gland and the tail vasculature were constricted by both adrenaline and noradrenaline. A flow reduction in the isolated preparations of about 10% at concentrations of adrenaline equalling that in normal cod plasma (30 nM) was increased to about 30% (tail) or 45% (gas gland) at concentrations of adrenaline similar to that found in cod plasma during stress (300 nM).The constrictory responses to adrenaline and noradrenaline were antagonized in all preparations by the alpha adrenoceptor antagonist, phentolamine (10–6 M) and the dilatory response of the branchial vasculature to these agonists was reduced or abolished by propranolol (10–6 M).It is concluded that adrenaline, in the concentrations found in cod plasma at rest and after stress, has pronounced effects on the organs studied, especially the gill vasculature, and may thus contribute to the overall control of the circulatory system. The branchial vasculature will also be affected by changes in noradrenaline concentration which occur in animals at rest and under stress.  相似文献   

7.
Divalent cation (Mn2+, Ca2+) entry into rat parotid acinar cells is stimulated by the release of Ca2+ from the internal agonist-sensitive Ca2+ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn2+ influx into internal Ca2+ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca2+-free medium for 10 min) and passive 45Ca2+ influx in basolateral membrane vesicles (BLMV). Mn2+ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn2+ entry appeared to be inactivated since it was not increased by raising extracellular [Mn2+] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn2+ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q10=1.7) at 21–37°C to 25 kcal/mol (Q10=3.0) at 21-8°C. Under the same conditions, Mn2+ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E a of 7.8 kcal/mol (Q10=1.5) and 6.2 kcal/mol (Q10 < 1.5), respectively. These data suggest that depletion-activated Mn2+ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn2+ entry into unstimulated acini.As in intact acini, Ca2+ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca2+ influx in BLMV at 37°C demonstrated the presence of two Ca2+ influx components: a saturable component, with K Ca =279 ± 43 m, Vmax = 3.38 ± 0.4 nmol Ca2+/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but Vmax decreased by 61% to 1.3 ± 0.4 nmol Ca2+/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca2+ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca2+ uptake at 37°C there was no significant loss of Ca2+ influx. These data suggest that the temperature sensitive high affinity Ca2+ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca2+ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.  相似文献   

8.
Summary Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond JCH coupling constants in proteins that are uniformly enriched with 13C. An empirical ,-surface is calculated which describes the deviation of 1JCH from its random coil value, using 203 1JCH values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which and are know with good precision from previous X-ray crystallographic studies. Residues in -helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in -sheet are small and, on average, slightly negative. Data indicate that 1JCH depends primarily on , and that 1JCH may be useful as a qualitative probe for secondary structure. Comparison of 1JCH coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the central helix is even more extensive than in free calmodulin. Supplementary material available from the authors: One table listing 352 1JCH and 1J-values, together with ,-values for 203 residues of known conformation. Two figures showing (a) a Ramachandran plot of the ,-values of 203 residues used in deriving 1J(,), and (b) the r.m.s.d. 1J(,) distribution.  相似文献   

9.
The regulation of total creatine content in a myoblast cell line   总被引:5,自引:0,他引:5  
Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na+]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na+,K+-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the 2-agonist clenbuterol and the cAMP analogue N6,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the 1 adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the 2 antagonist phentolamine; greater inhibition was caused by the 2 antagonist butoxamine than the 1 antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na+,K+-ATPase, we suggest that they stimulate Na+-creatine cotransport indirectly by increasing the transmembrane [Na+] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T3 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide  相似文献   

10.
Summary The conductance of the Ca2+-activated K+ channel (g K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V m ) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V m –E K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V m –E K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V m –E K) decreased from 20 towards zero mVg K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV m (–50 mV to 0). A further increase ing K(Ca) symmetrically aroundE K is suggested as (V m –E K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing K(Ca) from 50 to 70 S/cm2. Since the driving force (V m –E K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.  相似文献   

11.
Summary With the combined use of various two-dimensional (2D) NMR techniques, a complete assignment of the 1H and 13C resonances of oxytocin, , for two molecular states, protonated and unprotonated at the N-terminal group, was performed in dimethyl sulfoxide. A small but distinct change in the backbone conformation of the six-residue cyclic moiety, associated with the protonation, was first suggested from those NMR parameters relevant to conformation, such as change with temperature in the chemical shifts of the peptide amide protons and changes in chemical shifts and homonuclear as well as heteronuclear three-bond coupling constants. The solution structures of oxytocin for the protonated and unprotonated forms were then calculated using distance analysis in dihedral-angle space, based on a relaxation matrix evaluated from quantitative NOE intensities at different mixing times. Total amounts of 93 and 105 distances were determined for the protonated and the unprotonated forms, respectively. There were 25 interresidue distances relevant to the structure of the cyclic moiety for the protonated form of oxytocin and 43 for the unprotonated form. Overall structures with the lowest target penalty function were similar between the two forms, having a -turn structure at the endocyclic residues of the Tyr-Ile-Gln-Asn moiety. The local backbone conformations near the N-terminus, however, were significantly different between the two forms. This was found to be due to a change in the dihedral angle of the disulfide bridge (ss around C-S-S-C), which closes the ring in the cyclic peptide. The dihedral angle was about +90° for the unprotonated form and an intermediate value of about +45° for the protonated form.  相似文献   

12.
M. R. Davis 《Plant and Soil》1990,126(2):237-246
Concentrations of ions were measured in soil solutions from beech (Nothofagus) forests in remote areas of New Zealand and in solutions from beech (Fagus sylvatica) and Norway spruce (Picea abies) forests in North-East Bavaria, West Germany, to compare the chemistry of soil solutions which are unaffected by acid deposition (New Zealand) with those that are affected (West Germany). In New Zealand, soil solution SO4 2– concentrations ranged between <2 and 58 mol L–1, and NO3 concentrations ranged between <1 and 3 mol L–1. In West Germany, SO4 2– concentrations ranged between 80 and 700 mol L–1, and NO3 concentrations at three of six sites ranged between 39 and 3750 mol L–1, but was not detected at the remaining three sites. At all sites in New Zealand, and at sites where the soil base status was moderately high in West Germany, pH levels increased, and total Al (Alt) and inorganic monomeric Al (Ali) levels decreased rapidly with increasing soil depth. In contrast, at sites on soils of low base status in West Germany, pH levels increased only slightly, and Al levels did not decline with increasing soil depth.Under a high-elevation Norway spruce stand showing severe Mg deficiency and dieback symptoms in West Germany, soil solution Mg2+ levels ranged between 20 and 60 mol L, and were only half those under a healthy stand. Alt and Ali levels were substantially higher the healthy stand than under the unhealthy stand, indicating that Al toxicity was not the main cause of spruce decline.  相似文献   

13.
This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Cai Ca2+ inside vesicles - Ca0 Ca2+ outside vesicles - SR sarcoplasmic reticulum - PC phosphatidylcholine - PS phosphatidylserine - PG phosphatidylglycerol - PE phosphatidylethanolamine - DPH 1,6-diphenyl-1,3,5-hexatriene - n-AS n-(9-anthroyloxy) fatty acids - TMA-DPH 1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - -AR -adrenergic receptors - DHA dihydroalprenolol - AC adenylate cyclase - AC·Lca+– higher Ca2+ inside vesicles - AC·Lca– – lower Ca2+ on both side of vesicles - AC·Lca++ higher Ca2+ on both side of vesicles - AC·Lca– + higher Ca2+ outside vesicles - cAMP cyclic adenosine monophosphate - Gs stimulatory GTP-binding protein - GTP guanosine triposphate - GTPS guanosine 50-(3-thiotriphosphate)  相似文献   

14.
The chemical shift difference ([13C] – [13C]) is a reference-independent indicator of the Xaa-Pro peptide bond conformation. Based on a statistical analysis of the 13C chemical shifts of 1033 prolines from 304 proteins deposited in the BioMagRes database, a software tool was created to predict the probabilities for cis or trans conformations of Xaa-Pro peptide bonds. Using this approach, the conformation at a given Xaa-Pro bond can be identified in a simple NOE-independent way immediately after obtaining its NMR resonance assignments. This will allow subsequent structure calculations to be initiated using the correct polypeptide chain conformation.  相似文献   

15.
The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D2O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (Ri) of 3 the lamellar gel (L or Lc)-to-hexagonal type II (HII) phase sequence is observed on increasing the temperature. For Ri3 the thermotropic sequence (L or Lc)-L-HII is detected. On increasing hydration from Ri=3, the HII phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and HII phases are Ri 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the HII phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - Tc lamellar gel-to-lamellar fluid transition temperature - Th lamellar fluid-to-hexagonal transition temperature  相似文献   

16.
Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1000 U/mL collagenase for 3 h, at 37°C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates.The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence of collagenase treatment. In the absence of Mg2+ ions, the enzyme hydrolyzed PNPP with KM = 322.5 ± 15.3 M and V = 965.2 ± 45.8 U/mg, while in the presence of 2 mM Mg2+ ions, V increased 66%. Cobalt (K0.5 = 5.3 ± 0.3 M) and manganese (K0.5 = 0.72 ± 0.03 M) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg2+ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (KM = 105.1 ± 6.3 M and V = 64.9 ± 3.9 U/mg), but site-site interactions (nH = 1.2) were observed in the presence of 2 mM Mg2+ ions (V = 110.8 ± 5.5 U/mg; K0.5 = 42.7 ± 2.0 M).To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.  相似文献   

17.
Summary The conformation of chymotryptic fragment C2 of bacteriorhodopsin (residues 1–71) was studied by 2D1H NMR. The fragment was solubilized in a mixture of chloroform/methanol (11), 0.1 M LiClO4. Most of the resonances in1H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in -positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed -helical regions Pro8-Lys30 and Lys41-Leu62 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala.2-Gly6) adopts the helical conformation stabilized by 3 hydrogen bonds.  相似文献   

18.
Summary Surfactants, which provide a hydrophobic environment, may induce an ordered conformation in polypeptides and proteins that contain a sequence with helix- or -forming potential. This hypothesis has been illustrated in circular dichroic studies of oligopeptides and short polypeptides. These peptide-surfactant complexes can form (1) a helix, (2) a -form, (3) either form (depending on experimental conditions), or can remain in (4) an ordered form. The induced helix is stable in a surfactant solution below or above its critical micellar concentration, whereas the induced -form is usually converted back to an unordered form when the surfactant used is above its critical micellar concentration, or it is transformed into a helix in excess surfactant solution if the peptide has both the helix- and -forming potential. In most cases the observed conformations agree with those predicted from the amino acid sequences of the peptides. The induced conformation of a peptide can be destabilized by charges on the side groups having the same sign as that of surfactant ions. Disulfide bonds can inhibit the formation of induced conformation because of steric hindrance. The terminal effect can prevent a peptide from forming an ordered conformation near the NH2- and COOH-terminus.  相似文献   

19.
A new method to measure 1J(Ni,C i) and 2J(Ni,C (i – 1)) coupling constants in proteins based on a J-modulated sensitivity enhanced HSQC was introduced. Coupling constants were measured in the denatured and in the native state of ubiquitin and found to depend on the conformation of the protein backbone. Using a combined data set of experimental coupling constants from ubiquitin and staphylococcal nuclease (Delaglio et al., 1991), the angular dependence of the coupling constants on the backbone angles and was investigated. It was found that the size of 2J(Ni,C (i – 1)) correlates strongly with the backbone conformation, while only a weak conformational dependence on the size of 1J(Ni,C i) coupling constants was observed. Coupling constants in the denatured state of ubiquitin were uniform along the sequence of the protein and not dependent on a given residue type. Furthermore it was shown that the observed coupling constants were in good agreement with predicted coupling constants using a simple model for the random coil.  相似文献   

20.
A combination of stable isotope studies and 14Cdating were used to identify the main sources andprocesses controlling streamwater DOC and TIC in atemperate non-forested watershed. 13Cvalues for terrestrial (–24.9 to –29.1) and aquatic(–30.5 to –33.5) plants were similar to valuesreported in the literature for similar ecosystems.13C values for DOC in soil solution andstreamwater were consistent with soil and terrestrialvegetation, indicating that the terrestrial ecosystemis the dominant source of aquatic DOC in thiswatershed. 13C values of soil atmosphereCO2 (–17.2 to –25.2) were slightly lessnegative than would be expected for production viaaerobic soil microbial decomposition and rootrespiration. There was a close correspondence between13C values (–15.5 to –21.5) forstreamwater TIC and soil atmospheric CO2 in thecentral part of the catchment where the stream drainsCO2-rich peats. 14C dating showed thatalthough peat has been accumulating in the watershedfor at least 2700 years, DOC in soil pore water andstreamwater contains carbon of predominantly recentorigin (post-AD 1955).  相似文献   

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