New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as subtrates for various enzymes

Hiratsuka, Toshiaki

doi:10.1016/0167-4838(83)90267-4

Cited by 398 publications

(380 citation statements)

References 31 publications

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“…For a synthesis on the millimolar scale, the products were applied, after neutralization to about pH 7.5, to a column (2.5 x 20 cm) of QAE-Sephadex A-25, which was developed with a linear gradient of 0.35-0.5 M triethylammonium bicarbonate pH 7.5 over 4 1. The product was eluted at about 0.42 M. It was characterized by its ultraviolet spectrum, which was identical to that reported by Hiratsuka [24], and by alkaline hydrolysis to GDP. HPLC on C1 8 reversed-phase columns using an acetonitrile gradient in 50 mM phosphate pH 6 showed the presence of two products, namely the 2' and 3' isomers, in this preparation.…”

Section: Preparation Of N-methylanthraniloyl (Mant) Derivativessupporting

confidence: 76%

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Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction

Kalbitzer

Feuerstein

Goody

et al. 1990

European Journal of Biochemistry

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Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF‐Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)‐(Rp)‐[β‐17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line‐broadening of the EPR spectrum of Mn2+ at the active site of EF‐Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF‐Tu · GDP · Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2′(3′)‐O‐(N‐methylanthraniloyl)‐GDP is 6.2 × 10−3s−1 at 25°C and pH 6.8], we conclude that the broadened signal arises from the EF‐Tu · Mn · GDP · Pi complex, the predominant steady‐state species. During the hydrolysis of GTP the Mn2− remains bound to the β‐phosphate oxygen of GDP which arises from the βpro‐S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF‐Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)‐[β‐17O]GTP in the corresponding complex in the presence of EF‐Ts.

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Section: Preparation Of N-methylanthraniloyl (Mant) Derivativessupporting

confidence: 76%

“…of guanine nucleotides mant-GDP was prepared by reaction of methylisatoic anhydride with GDP essentially as described by Hiratsuka [24]. The mixture of products was separated not as described by Hiratsuka, by gel filtration, but by ion-exchange chromatography on QAE-Sephadex.…”

Section: Preparation Of N-methylanthraniloyl (Mant) Derivativesmentioning

confidence: 99%

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction

Kalbitzer

Feuerstein

Goody

et al. 1990

European Journal of Biochemistry

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“…ADP and ATP concentrations were determined spectrophotometrically before each experiment by absorbance at 259 nm, ε 259 = 15,400 M -1 cm -1 . MantATP concentrations were determined by absorbance at 255 nm, ε 255 = 23,300 M -1 cm -1 (17).…”

Section: Reagents Proteins and Buffersmentioning

confidence: 99%

Temperature Dependence of Nucleotide Association and Kinetic Characterization of Myo1b

et al. 2006

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Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 °C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide inaccessible state to a nucleotide accessible state (kalpha). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3 fold greater than other characterized myosins (e.g, myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (V max and K ATPase ). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to kalpha suggesting a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.Myosin-Is are the single-headed, low-molecular-weight members of the myosin superfamily that are proposed to link cellular membranes with the actin cytoskeleton. Myosin-I isoforms bind phosphoinositides directly (1) and function in several important cellular processes, including membrane retraction, macropinocytosis, phagocytosis, membrane trafficking, cellcell adhesion, and mechanical signal transduction (2-8).The biochemical mechanisms of long-tail and short-tail myosin-I isoforms have been studied (9-12), with myo1b being the best characterized short-tail isoform (13)(14)(15). The myo1b ATPase mechanism (Scheme 1) is notable in that (a) the maximum rate of ATP binding and population of the myo1b IQ weakly-bound states is > 30-fold slower than myosin-II, (b) nucleotide-free myo1b is in equilibrium between a state that binds nucleotide (AM′) and a state that does not bind nucleotide (AM), (c) the rate of transition between AM and AM′ (k +α ) states is similar to the rate of ADP release (k +5 ′), and (d) ADP release is slow and is accompanied by a rotation of the lever arm.We proposed that the strikingly slow actomyo1b ATPase rate constants are a property of all short-tail myosin-I isoforms (11,16). However, it has been shown recently that short-tailed Dictyostelium myosin-IE (not to be confused with vertebrate long-tail myo1e (11)) has kinetic rate constants that are substantially faster than vertebrate short-tail isoforms (12). Because of † EMO was supported by grants from the National Institutes of Health (GM57247 and AR051174). JHL was supported by a training grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR053461). *Corresponding author: E. Michael Ostap, Department of Physiology, University of Pe...

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“…pM). Thus, mantATP seems to be as good an ATP analogue for the ncd motor domain as it is for kinesin (Sadhu & Taylor, 1992), myosin (Hiratsuka, 1983), and dynein (Inaba et al, 1989). Hence, we were able to utilize mantATP for a kinetic characterization of the ncd motor domain ATPase cycle.…”

Section: Concentration Of Nacl (M)mentioning

confidence: 99%

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Shimizu¹,

Sablin²,

Vale³

et al. 1995

Biochemistry

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ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose chromatography, and trace amounts of contaminants were removed by passing through a MonoQ column. The yield was 20 mg from a 500 mL. culture of E. coli. The purified ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275 nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive ATP, 3H at adenine but not 32P at y-phosphate was retained by the protein on gel filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding to the ncd motor domain is tight, although there is an equilibrium between the protein and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the absence of microtubules, but microtubules activated the turnover to a similar extent to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of mantATP increased at 0.005 s-l, which is likely to reflect the release of endogenous ADP and incorporation of mantATP into the protein. The fluorescence intensity of the ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing the mantADP release. The rate was accelerated more than 1000-fold to 3.3 s-l by the presence of saturating microtubules. The profiles of the mantADP release rate and the mantATP turnover rate versus microtubule concentration were nearly identical, except that the maximal rate of mantATP turnover was 30% lower than the maximal mantADP release rate. This result suggests that mantADP release substantially contributes to the overall cycle time. We have also measured the equilibrium dissociation constants for ncd motor domain binding to microtubules in the presence of ADP [&(ADP) = 4-5 pM] and ATP [&(ATP) = 6-7.5 pM] and the apparent halfmaximal microtubule stimulation of ATPase activity (5-7 pM) under an identical condition. The enzymatic and microtubule-binding characteristics of ncd motor domain reported here are similar to those of kinesin. The notable exception, however, is that &(ADP) for kinesin is 2-3-fold larger than &(ATP), which suggests the existence of a weak binding ADP state in the case of kinesin but not in the case of ncd motor domain. Such a difference could be relevant for understanding the opposite polarity of movement of these two microtubule motors.Eukaryotes generate cytoplasmic motility using two types of filaments, actin and microtubules. Actin-based motility is driven by motors that belong to the myosin superfamily

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New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as subtrates for various enzymes

Cited by 398 publications

References 31 publications

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction

Temperature Dependence of Nucleotide Association and Kinetic Characterization of Myo1b

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

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New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as subtrates for various enzymes

Cited by 398 publications

References 31 publications

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O–55Mn superhyperfine interaction

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O–55Mn superhyperfine interaction

Temperature Dependence of Nucleotide Association and Kinetic Characterization of Myo1b

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

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Product

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Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the ¹⁷O–⁵⁵Mn superhyperfine interaction