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1.
Carbon fiber running-specific prostheses have allowed lower extremity amputees to participate in running activity by providing spring-like properties in their affected limb. It has been established that as running speed increases, stiffness of the leg spring (leg stiffness; kleg) remains constant in non-amputees. Although a better understanding of kleg regulation may be helpful for the development of spring-based prostheses, little is known about stiffness regulation in unilateral transfemoral amputees. The aim of this study was to investigate stiffness regulation at different running speeds in unilateral transfemoral amputees wearing a running-specific prosthesis. Nine unilateral transfemoral amputees performed running on an instrumented treadmill across a range of speeds (30, 40, 50, 60, and 70% of their maximum running speed). Using a spring-mass model, kleg was calculated as the ratio of maximal vertical ground reaction force to maximum leg compression during the stance phase in both affected and unaffected limbs. We found a decrease in kleg from the slower speed to 70% speed for the affected limb, whereas no change was present in the unaffected limb. Specifically, there was a significant differences in the kleg between 30% and 70%, 40% and 70%, and 50% and 70%, and the magnitude of the kleg difference between affected and unaffected limbs varied with variations in running speeds in unilateral TFAs with an RSP. These results suggest the kleg regulation strategy of unilateral transfemoral amputees is not the same in the affected and unaffected limbs across a range of running speeds. 相似文献
2.
Hiroaki Hobara Brian S. Baum Hyun-Joon Kwon Ross H. Miller Toru Ogata Yoon Hyuk Kim Jae Kun Shim 《Journal of biomechanics》2013
Carbon fiber running-specific prostheses (RSPs) have allowed individuals with lower extremity amputation (ILEA) to participate in running. It has been established that as running speed increases, leg stiffness (Kleg) remains constant while vertical stiffness (Kvert) increases in able-bodied runners. The Kvert further depends on a combination of the torsional stiffnesses of the joints (joint stiffness; Kjoint) and the touchdown joint angles. Thus, an increased understanding of spring-like leg function and stiffness regulation in ILEA runners using RSPs is expected to aid in prosthetic design and rehabilitation strategies. The aim of this study was to investigate stiffness regulation to various overground running speeds in ILEA wearing RSPs. Eight ILEA performed overground running at a range of running speeds. Kleg, Kvert and Kjoint were calculated from kinetic and kinematic data in both the intact and prosthetic limbs. Kleg and Kvert in both the limbs remained constant when running speed increased, while intact limbs in ILEA running with RSPs have a higher Kleg and Kvert than residual limbs. There were no significant differences in Kankle, Kknee and touchdown knee angle between the legs at all running speeds. Hip joints in both the legs did not demonstrate spring-like function; however, distinct impact peaks were observed only in the intact leg hip extension moment at the early stance phase, indicating that differences in Kvert between limbs in ILEA are due to attenuating shock with the hip joint. Therefore, these results suggest that ILEA using RSPs has a different stiffness regulation between the intact and prosthetic limbs during running. 相似文献
3.
Ferris, Daniel P., and Claire T. Farley. Interaction ofleg stiffness and surface stiffness during human hopping.J. Appl.Physiol. 82(1): 15-22, 1997.When mammals run,the overall musculoskeletal system behaves as a single linear "legspring." We used force platform and kinematic measurements todetermine whether leg spring stiffness(kleg) isadjusted to accommodate changes in surface stiffness(ksurf) whenhumans hop in place, a good experimental model for examiningadjustments tokleg in bouncinggaits. We found thatkleg was greatlyincreased to accommodate surfaces of lower stiffnesses. The seriescombination ofkleg andksurf[total stiffness(ktot)]was independent ofksurf at a givenhopping frequency. For example, when humans hopped at a frequency of 2 Hz, they tripled theirkleg on the leaststiff surface(ksurf = 26.1 kN/m; kleg = 53.3 kN/m) compared with the most stiff surface(ksurf = 35,000 kN/m; kleg = 17.8 kN/m). Values forktot were notsignificantly different on the least stiff surface (16.7 kN/m) and themost stiff surface (17.8 kN/m). Because of thekleg adjustment,many aspects of the hopping mechanics (e.g., ground-contact time andcenter of mass vertical displacement) remained remarkably similardespite a >1,000-fold change inksurf. This studyprovides insight into howkleg adjustmentscan allow similar locomotion mechanics on the variety of terrainsencountered by runners in the natural world. 相似文献
4.
The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F0) and closed (FM) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, FV = FM − F0, over FM, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the FV/FM ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of FV/FM principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F0 and FM indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo. 相似文献
5.
Sinisa Bjelic Yakov Kipnis Ling Wang Zbigniew Pianowski Sergey Vorobiev Min Su Jayaraman Seetharaman Rong Xiao Gregory Kornhaber John F. Hunt Liang Tong Donald Hilvert David Baker 《Journal of molecular biology》2014
Designed retroaldolases have utilized a nucleophilic lysine to promote carbon–carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (> 10− 3 s− 1) and kcat/KM (11–25 M− 1 s− 1) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the > 105-fold rate accelerations that were achieved are within 1–3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat = 106 to 108) and an extensively evolved computational design (kcat/kuncat > 107). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches. 相似文献
6.
Valentina Bosello-Travain Marcus Conrad Giorgio Cozza Alessandro Negro Silvia Quartesan Monica Rossetto Antonella Roveri Stefano Toppo Fulvio Ursini Mattia Zaccarin Matilde Maiorino 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Methods
Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Results
Oxidation of the CP is fast (k+ 1 > 103 M− 1 s− 1), however the rate of reduction by GSH is slow (k′+ 2 = 12.6 M− 1 s− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+ 1 > 103 M− 1 s− 1), but not by Trx. By surface plasmon resonance analysis, a KD = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.Conclusions
GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significance
In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. 相似文献7.
ADP-ribosyl cyclase and NAD+ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide mononucleotide (NMN+) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (kcat/KM = 0.9-10 μM−1 s−1) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the kcat/KM values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM−1 s−1). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN+, kcat/KM = 0.05-0.4 μM−1 s−1. The kcat/KM values for the exchange reaction with NMN+ were generally similar (0.04-0.12 μM−1 s−1) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The kcat/KM values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38. 相似文献
8.
Michael J. Vasbinder 《Inorganica chimica acta》2008,361(11):3193-3198
Oxidation of the title complexes with ozone takes place by hydrogen atom, hydride, and electron transfer mechanisms. The reaction with (NH3)4(H2O)RhH2+ is a two electron process, believed to involve hydride transfer with a rate constant k = (2.2 ± 0.2) × 105 M−1 s−1 and an isotope effect kH/kD = 2. The oxidation of (NH3)4(H2O)RhOOH2+ to (NH3)4(H2O)RhOO2+ by an apparent hydrogen atom transfer is quantitative and fast, k = (6.9 ± 0.3) × 103 M−1 s−1, and constitutes a useful route for the preparation of the superoxo complex. The latter is also oxidized by ozone, but more slowly, k = 480 ± 50 M−1 s−1. 相似文献
9.
The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10−13 M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k1) of about 2 × 10−3 s−1 (by deduction k−1 is about10−4 s−1) and assembles into the active dimeric state with a bimolecular rate constant (k2) of about 2 × 104 M−1 s−1. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k−2 ∼ 3 × 10−7 s−1). 相似文献
10.
Qurra-tul-Ann Afza Gardner Hooria Younas Muhammad Akhtar 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):182-190
Human M-proinsulin was cleaved by trypsin at the R31R32–E33 and K64R65–G66 bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K59R60–G61 bond but at the B/C junction cleavage occurred at the R31R32–E33 as well as the R31–R32E33 bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the kcat./Km values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 105 s− 1 M− 1) and the cleavage of the K29–T30 bond of M-insulin-RR (1.3 ± 0.07 × 105 s− 1 M− 1). However, the K29–T30 bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (kcat./Km values around 1000 s− 1 M− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K29–T30 bond becomes shielded, exposed then shielded again respectively. 相似文献
11.
Daria Petzhold Burcu Simsek Ralf Meißner Shokoufeh Mahmoodzadeh Ingo Morano 《Biochemical and biophysical research communications》2014
Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actinala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (KD = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (kon: 1018 M−1 s−1) compared with kon of the hVLC-1/α-actin complex interaction (2908 M−1 s−1). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions. 相似文献
12.
Rücker's walk count (WC) indices are well-known topological indices (TIs) used in Chemoinformatics to quantify the molecular structure of drugs represented by a graph in Quantitative structure–activity/property relationship (QSAR/QSPR) studies. In this work, we introduce for the first time the higher-order (kth order) analogues (WCk) of these indices using Markov chains. In addition, we report new QSPR models for large complex networks of different Bio-Systems useful in Parasitology and Neuroinformatics. The new type of QSPR models can be used for model checking to calculate numerical scores S(Lij) for links Lij (checking or re-evaluation of network connectivity) in large networks of all these fields. The method may be summarized as follows: (i) first, the WCk(j) values are calculated for all jth nodes in a complex network already created; (ii) A linear discriminant analysis (LDA) is used to seek a linear equation that discriminates connected or linked (Lij = 1) pairs of nodes experimentally confirmed from non-linked ones (Lij = 0); (iii) The new model is validated with external series of pairs of nodes; (iv) The equation obtained is used to re-evaluate the connectivity quality of the network, connecting/disconnecting nodes based on the quality scores calculated with the new connectivity function. The linear QSPR models obtained yielded the following results in terms of overall test accuracy for re-construction of complex networks of different Bio-Systems: parasite–host networks (93.14%), NW Spain fasciolosis spreading networks (71.42/70.18%) and CoCoMac Brain Cortex co-activation network (86.40%). Thus, this work can contribute to the computational re-evaluation or model checking of connectivity (collation) in complex systems of any science field. 相似文献
13.
Immacolata Del Giudice Danila Limauro Emilia Pedone Simonetta Bartolucci Gabriella Fiorentino 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2071-2079
Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2 × 104 M− 1 s− 1. It also exhibited weak phosphatase activity with a kcat/KM value of 2.7 × 10− 4 M− 1 s− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase. 相似文献
14.
GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (kcat/KM(glucose) = 93 μM−1 s−1) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (kcat/KM(glucose) = 27 μM−1 s−1). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FMox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FMox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(kcat/KM(FMox) = 27 μM−1 s−1 and 17 μM−1 s−1 respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions. 相似文献
15.
Yi-Pin Lin WeiWei Yan Yogendra Sharma Yung-Fu Chang 《Biochemical and biophysical research communications》2009,389(1):57-7386
Infection by pathogenic strains of Leptospira hinges on the pathogen’s ability to adhere to host cells via extracellular matrix such as fibronectin (Fn). Previously, the immunoglobulin-like domains of Leptospira Lig proteins were recognized as adhesins binding to N-terminal domain (NTD) and gelatin binding domain (GBD) of Fn. In this study, we identified another Fn-binding motif on the C-terminus of the Leptospira adhesin LigB (LigBCtv), residues 1708-1712 containing sequence LIPAD with a β-strand and nascent helical structure. This motif binds to 15th type III modules (15F3) (KD = 10.70 μM), and association (kon = 600 M−1 s−1) and dissociation (koff = 0.0129 s−1) rate constants represents a slow binding kinetics in this interaction. Moreover, pretreatment of MDCK cells with LigB1706-1716 blocked the binding of Leptospira by 39%, demonstrating a significant role of LigB1706-1716 in cellular adhesion. These data indicate that the LIPAD residues (LigB1708-1712) of the Leptospira interrogans LigB protein bind 15F3 of Fn at a novel binding site, and this interaction contributes to adhesion to host cells. 相似文献
16.
The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2–11 and at temperature 90 °C for 2 1/2 h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE .The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% α-helix, 49% β-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin–API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC50 and Ki values 4.0 nM and (3.83 nM–5.31 nM) respectively. The overall inhibition constant Ki? value is 0.107 ± 0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I ? (k4, k5) EI ? (k6, k7) EI?. Rate constant k6 = 2.73 ± 0.32 s− 1 reveals a fast isomerization of enzyme–inhibitor complex and very slow dissociation as proved by k7 = 0.068 ± 0.009 s− 1. The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI?. 相似文献
17.
Forced unbinding of complementary macromolecules such as ligand-receptor complexes can reveal energetic and kinetic details governing physiological processes ranging from cellular adhesion to drug metabolism. Although molecular-level experiments have enabled sampling of individual ligand-receptor complex dissociation events, disparities in measured unbinding force FR among these methods lead to marked variation in inferred binding energetics and kinetics at equilibrium. These discrepancies are documented for even the ubiquitous ligand-receptor pair, biotin-streptavidin. We investigated these disparities and examined atomic-level unbinding trajectories via steered molecular dynamics simulations, as well as via molecular force spectroscopy experiments on biotin-streptavidin. In addition to the well-known loading rate dependence of FR predicted by Bell's model, we find that experimentally accessible parameters such as the effective stiffness of the force transducer k can significantly perturb the energy landscape and the apparent unbinding force of the complex for sufficiently stiff force transducers. Additionally, at least 20% variation in unbinding force can be attributed to minute differences in initial atomic positions among energetically and structurally comparable complexes. For force transducers typical of molecular force spectroscopy experiments and atomistic simulations, this energy barrier perturbation results in extrapolated energetic and kinetic parameters of the complex that depend strongly on k. We present a model that explicitly includes the effect of k on apparent unbinding force of the ligand-receptor complex, and demonstrate that this correction enables prediction of unbinding distances and dissociation rates that are decoupled from the stiffness of actual or simulated molecular linkers. 相似文献
18.
The binding affinity of the two substrate–water molecules to the water-oxidizing Mn4CaO5 catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S2 and S3 states by the exchange of bound 16O-substrate against 18O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: kf = 52 ± 8 s− 1 and ks = 1.9 ± 0.3 s− 1 in the S2 state, and kf = 42 ± 2 s− 1 and kslow = 1.2 ± 0.3 s− 1 in S3, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S2 state, which confirms beyond doubt that both substrate–water molecules are already bound in the S2 state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S2 → S3 transition. Implications for recent models for water-oxidation are briefly discussed. 相似文献
19.
Two 15N-labelled cis-Pt(II) diamine complexes with dimethylamine (15N-dma) and isopropylamine (15N-ipa) ligands have been prepared and characterised. [1H,15N] HSQC NMR spectroscopy is used to obtain the rate and equilibrium constants for the aquation of cis-[PtCl2(15N-dma)2] at 298 K in 0.1 M NaClO4 and to determine the pKa values of cis-[PtCl(H2O)(15N-dma)2]+ (6.37) and cis-[Pt(H2O)2(15N-dma)2]2+ (pKa1 = 5.17, pKa2 = 6.47). The rate constants for the first and second aquation steps (k1 = (2.12 ± 0.01) × 10−5 s−1, k2 = (8.7 ± 0.7) × 10−6 s−1) and anation steps (k−1 = (6.7 ± 0.8) × 10−3 M−1 s−1, k−2 = 0.043 ± 0.004 M−1 s−1) are very similar to those reported for cisplatin under similar conditions, and a minor difference is that slow formation of the hydroxo-bridged dimer is observed. Aquation studies of cis-[PtCl2(15N-ipa)2] were precluded by the close proximity of the NH proton signal to the 1H2O resonance. 相似文献
20.
Svetlana Sharoyan Alvard Antonyan Sona Mardanyan Hayk Harutyunyan Naira Movsisyan Narek Hovnanyan Karlen Hovnanyan 《Neurochemistry international》2013
The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (Km = 37.5 μM, kcat/Km = 1.7 × 103 M−1sec−1) and Aβ42 (Km = 138.4 μM, kcat/Km = 1.90 × 102 M−1sec−1). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates. 相似文献