[12] Myosin active-site trapping with vanadate ion

Goodno, Charles C.

doi:10.1016/0076-6879(82)85014-3

Cited by 193 publications

(172 citation statements)

References 6 publications

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“…The Vi was prepared as detailed by Goodno [25], and the Vi concentration of stock solutions determined by molar absorbance (λ 268nm = 3600 M -1 ). After incubation for 4 min at 37 °C to allow for nucleotide binding and trapping, 10 mM MgATP (pH 7.5) was added to reduce non-specific binding.…”

Section: Az-atp Binding and Vi-trappingmentioning

confidence: 99%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMPactivated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α 32 P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His 10 -tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (V Max of 58 ± 5 nmol/min/mg protein, K M (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTRdependent transport of 125 I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α 32 P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the cAMP-activated chloride channel encoded by the gene defective in patients with the disease [1,2]. As a member of the ATP-Binding Cassette (ABC) superfamily of membrane transporters, CFTR shares a conserved architecture consisting of two homologous halves, each containing a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion ...

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Section: Az-atp Binding and Vi-trappingmentioning

confidence: 99%

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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“…Vanadate inhibits myosin ATPase activity through the formation, in the absence of actin, of a long-lived complex with MgADP that is believed to mimic the transition state of myosin**-ADP-Pi [6]. In the presence of actin, the rate of vanadate release increases by 10 5 compared to that of myosin.ADP.V 1 alone (5 · 10 À6 s À1 ), and consequently the inhibition is reverted [7]. Nevertheless, it has been described that as long as excess Vi is present (denoting a mixture of monomeric and polymeric vanadate ions), the formation of the myosin.ADP.V 1 complex is favoured even with the ''destabilizing'' influence of actin [8,9].…”

Section: Introductionmentioning

confidence: 99%

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation

Tiago

Aureliano

Moura

2004

Journal of Inorganic Biochemistry

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Recently reported decameric vanadate (V 10 ) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V 10 species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta-and decavanadate solutions, indicating that V 10 and tetrameric vanadate (V 4 ) have the same binding sites in myosin S1. Concentrations as low as 50 lM decavanadate (5 lM V 10 species) induces 30% of protein cleavage, whereas 500 lM metavanadate is needed to attain the same extent of cleavage. After irradiation, V 10 species is rapidly decomposed, upon protein addition, forming vanadyl (V 4+ ) species during the process. It was also observed by NMR line broadening experiments that, V 10 competes with V 4 for the myosin S1 binding sites, having a higher affinity. In addition, V 4 interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V 10 appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V 10 .

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“…Concentration of Sl was determined spectrophotometrically by using E$, nm = 7.5. The Sl ADP Vi complex was obtained by incubation of Sl (1 mg/ml) with 1 mM MgCl,, 0.2 mM ADP and 0.2 mM Vi [5] for 30 min at 20°C in a medium containing 30 mM HEPES, pH 7.3. In order to obtain the Sl ADP BeF; complex, Sl (1 mg/ml) was incubated for 5 min at 20°C in 1 mM MgCl,, 30 mM HEPES, pH 7.3,0.2 mM ADP, and 5 mM NaF; after addition of 0.2 mM BeCl, the reaction mixture was further incubated at 20°C for 25 min [7].…”

Section: Methodsmentioning

confidence: 99%

Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride

et al. 1993

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The thermal unfolding of the myosin subfragment 1 (Sl) in its stable complex with ADP and beryllium fluoride (Sl . ADP . BeF;) was studied by differential scanning calorimetry. It has been shown that the structure of the Sl molecule in the Sl ADP . BeF; complex is similar to that of Sl in its complex with ADP and orthovanadate (Sl ADP . Vi) but differs radically from that of nucleotide-free Sl and Sl in the Sl . ADP complex. It is concluded that the Sl . ADP . BeF; complex can be considered, like the Sl . ADP . Vi complex, a stable structural analogue of the myosin head ADP Pi transition state of the myosin-catalyzed ATP hydrolysis.

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[12] Myosin active-site trapping with vanadate ion

Cited by 193 publications

References 6 publications

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation

Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride

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