Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.     

Assay for the determination of urinary 8-hydroxy-2′-deoxyguanosine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic procedure with electrochemical detection is described for the determination of urinary 8-hydroxy-2′-deoxyguanosine, a major oxidative DNA lesion induced by radical forming agents. A two-step solid-phase extraction procedure was followed for extracting 8-hydroxy-2′-deoxyguanosine from human urine and the analysis was performed on a RP-18 analytical column under isocratic conditions. The limit of detection of 8-hydroxy-2′-deoxyguanosine in urine was found to be 0.9 nM. The non-invasive assay provides an indirect measurement of oxidative DNA damage.     

Phosphodiesterase inhibitors. Part 1: Synthesis and structure-activity relationships of pyrazolopyridine-pyridazinone PDE inhibitors developed from ibudilast

Ibudilast [1-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-2-methylpropan-1-one] is a nonselective phosphodiesterase inhibitor used clinically to treat asthma. Efforts to selectively develop the PDE3- and PDE4-inhibitory activity of ibudilast led to replacement of the isopropyl ketone by a pyridazinone heterocycle. Structure-activity relationship exploration in the resulting 6-(pyrazolo[1,5-a]pyridin-3-yl)pyridazin-3(2H)-ones revealed that the pyridazinone lactam functionality is a critical determinant for PDE3-inhibitory activity, with the nitrogen preferably unsubstituted. PDE4 inhibition is strongly promoted by introduction of a hydrophobic substituent at the pyridazinone N(2) centre and a methoxy group at C-7′ in the pyrazolopyridine. Migration of the pyridazinone ring connection from the pyrazolopyridine 3′-centre to C-4′ strongly enhances PDE4 inhibition. These studies establish a basis for development of potent PDE4-selective and dual PDE3/4-selective inhibitors derived from ibudilast.     

Phosphodiesterase inhibitors. Part 2: Design, synthesis, and structure-activity relationships of dual PDE3/4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory and bronchodilatory activity

A structural survey of pyrazolopyridine-pyridazinone phosphodiesterase (PDE) inhibitors was made with a view to optimization of their dual PDE3/4-inhibitory activity for respiratory disease applications. These studies identified (−)-6-(7-methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridine-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490, compound 2ac) as a compound with potent combined bronchodilatory and anti-inflammatory activity and an improved therapeutic window over roflumilast.     

Anionic effects on the formation of tridentate 4′-chloro-2,2′:6′,2″-terpyridine copper(II) complexes having 1:1 or 1:2 ratio of metal and ligand and their crystal symmetry

A facile synthetic procedure has been used to prepare one five-coordinate and four six-coordinate copper(II) complexes of 4′-chloro-2,2′:6′,2″-terpyridine (tpyCl) ligand with different counterions (, , , , and ) in high yields. They are formulated as [Cu(tpyCl-κ³N,N′,N′′)(SO₄-κO)(H₂O-κO)] · 2H₂O (1), trans-[Cu(tpyCl-κ³N,N′,N″)(NO₃-κO)₂(H₂O-κO)] (2), [Cu(tpyCl-κ³N,N′,N″)₂](BF₄)₂ (3), [Cu(tpyCl-κ³N,N′,N″)₂](PF₆)₂ (4) and [Cu(tpyCl-κ³N,N′,N″)₂](ClO₄)₂ (5) and versatile interactions in supramolecular level including coordinative bonding, O-H?O, O-H?Cl, C-H?F, and C-H?Cl hydrogen bonding, π-π stacking play essential roles in forming different frameworks of 1-5. It is concluded that the difference of coordination abilities of the counterions used and the experimental conditions codominate the resulting complexes with 1:1 or 1:2 ratio of metal and ligand.     

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3

Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg²⁺, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.     

Non-natural nucleotides as probes for the mechanism and fidelity of DNA polymerases

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides.     

Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O₂, but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O₂ sensing responsible for the regulation of the enzymatic activity.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

Ribonuclease A homologues of the zebrafish: polymorphism, crystal structures of two representatives and their evolutionary implications

The widespread and functionally varied members of the ribonuclease A (RNase A) superfamily provide an excellent opportunity to study evolutionary forces at work on a conserved protein scaffold. Representatives from the zebrafish are of particular interest as the evolutionary distance from non-ichthyic homologues is large. We conducted an exhaustive survey of available zebrafish DNA sequences and found significant polymorphism among its four known homologues. In an extension of previous nomenclature, the variants have been named RNases ZF-1a-c,-2a-d,-3a-e and-4. We present the first X-ray crystal structures of zebrafish ribonucleases, RNases ZF-1a and-3e at 1.35-and 1.85 Å resolution, respectively. Structure-based clustering with ten other ribonuclease structures indicates greatest similarity to mammalian angiogenins and amphibian ribonucleases, and supports the view that all present-day ribonucleases evolved from a progenitor with three disulphide bonds. In their details, the two structures are intriguing melting-pots of features present in ribonucleases from other vertebrate classes. Whereas in RNase ZF-1a the active site is obstructed by the C-terminal segment (as observed in angiogenin), in RNase ZF-3e the same region is open (as observed in more catalytically efficient homologues). The progenitor of present-day ribonucleases is more likely to have had an obstructive C terminus, and the relatively high similarity (late divergence) of RNases ZF-1 and-3 infers that the active site unblocking event has happened independently in different vertebrate lineages.     

Biomimetic conversion of (-)-fusoxypyridone and (-)-oxysporidinone to (-)-sambutoxin: further evidence for the structure of the tricyclic pyridone alkaloid, (-)-fusoxypyridone

Biomimetic-type reactions of the tricyclic pyridone alkaloid, (−)-fusoxypyridone [(−)-4,6′-anhydrooxysporidinone] (1), recently encountered in an endophytic strain of Fusarium oxysporum, and (−)-oxysporidinone (2) afforded (−)-sambutoxin (3) and an analogue of 1, identified as (−)-1′(6′)-dehydro-4,6′-anhydrooxysporidinone (4), thus confirming the structure previously proposed for 1 and suggesting that 1-3 bear the same relative stereochemistry. Oxidation of 4 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) yielded a hitherto unknown sambutoxin analogue, (−)-4,2′-anhydrosambutoxin (5).     

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

利用AFLP遗传连锁图定位大麦苗期对叶锈病的部分抗性基因

借助大麦染色体ＡＦＬＰ标记遗传连锁图和ＭａｐＱＴＬＶ３．０作图软件,对大麦叶病的数量抗性基因进行了定位分析,明确了大麦部分抗性品种Ｖａｄａ对叶锈病的潜育期由分别位于染色体１、２、６、７上离短臂末端７９ｃＭ、１８６ｃＭ、５８ｃＭ和１１７ｃＭ处的４个数量抗性基因所控制。     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Monogalactosyl diacylglycerol,a replicative DNA polymerase inhibitor,from spinach enhances the anti-cell proliferation effect of gemcitabine in human pancreatic cancer cells

Background

Gemcitabine (GEM) is used to treat various carcinomas and represents an advance in pancreatic cancer treatment. In the screening for DNA polymerase (pol) inhibitors, a glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), was isolated from spinach.

Methods

Phosphorylated GEM derivatives were chemically synthesized. In vitro pol assay was performed according to our established methods. Cell viability was measured using MTT assay.

Results

Phosphorylated GEMs inhibition of mammalian pol activities assessed, with the order of their effect ranked as: GEM-5′-triphosphate (GEM-TP) > GEM-5′-diphosphate > GEM-5′-monophosphate > GEM. GEM suppressed growth in the human pancreatic cancer cell lines BxPC-3, MIAPaCa2 and PANC-1 although phosphorylated GEMs showed no effect. MGDG suppressed growth in these cell lines based on its selective inhibition of replicative pol species. Kinetic analysis showed that GEM-TP was a competitive inhibitor of pol α activity with nucleotide substrates, and MGDG was a noncompetitive inhibitor with nucleotide substrates. GEM combined with MGDG treatments revealed synergistic effects on the inhibition of DNA replicative pols α and γ activities compared with GEM or MGDG alone. In cell growth suppression by GEM, pre-addition of MGDG significantly enhanced cell proliferation suppression, and the combination of these compounds was found to induce apoptosis. In contrast, GEM-treated cells followed by MGDG addition did not influence cell growth.

Conclusions

GEM/MGDG enhanced the growth suppression of cells based on the inhibition of pol activities.

General significance

Spinach MGDG has great potential for development as an anticancer food compound and could be an effective clinical anticancer chemotherapy in combination with GEM.     

Fluorescent labeling of a carboxyl group of sialic acid for MALDI-MS analysis of sialyloligosaccharides and ganglioside

We developed a modified method enabling stable MALDI-MS analysis and fluorescent detection of sialyl-compounds. The modification involved the amidation of sialic acid (Neu5Ac) at the position of the carboxyl group using the fluorescent reagent, 2-(2-pyridilamino)ethylamine (PAEA). In this study the following sialyl-compounds were amidated, 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), and ganglioside GM3. Yields of PAEA-3′-SL, PAEA-6′-SL, and PAEA-GM3 were 45%, 60%, and 30%, respectively. The PAEA-amidation enabled fluorescence detection of structural isomers using HPLC and TLC at sensitivity levels as low as pmol. In MALDI-TOF-MS and/or MS/MS analysis in positive ion mode, PAEA-amidation provided the following advantages: suppression of preferential cleavage of Neu5Ac; enhancement of molecular-related ion intensities; simplification of MS spectra; and finally, since PAEA-amidation did not cleave the linkage between sugar and aglycon of sialylglycoconjugate, MALDI-TOF-MS and MS/MS analyses revealed the complete structure of the molecule.     

Nucleotide-Induced Flexibility Change in Neck Linkers of Dimeric Kinesin as Detected by Distance Measurements Using Spin-Labeling EPR

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼ 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered.We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.