Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography

Agustoni, Elia; Teixeira, Raphael Dias; Huber, Markus; Flister, Susanne; Hiller, Sebastian; Schirmer, Tilman

doi:10.1016/j.ab.2021.114523

Cited by 3 publications

(3 citation statements)

References 13 publications

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“…The ATPase activity of BiP NBD was determined under single-turnover conditions as previously described with minor modi cations of the protocol 44 . The main-modi cation came to replace the radioactivitybased detection by a label free anion-exchange based quanti cation 57 . The BiP NBD-ATP complex was formed by mixing BiP NBD with 10 mM ATP in formation buffer (25 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl 2 ) and incubating the mixture for 5 min on ice.…”

Section: Single Turn-over Assaymentioning

confidence: 99%

“…Next, the complex was separated from unbound nucleotide by gel ltration columns and then incubated at 37°C for variable time, until the reaction was stopped by addition of HCl. From the resulting samples, the [ATP]/[ADP] ratio was determined by anion exchange chromatography 57 . The data were tted to a monoexponential, which corresponds to the ATP half-life time (Fig.…”

Section: Combined Measurement Of the Functional Cycle Kinetic Parametersmentioning

confidence: 99%

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Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain

Hiller,

Mas

2024

Preprint

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The 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for protein biogenesis and homeostasis in all kingdoms of life. Hsp70 activity is driven by ATP hydrolysis in the nucleotide binding domain (NBD). Here, we report an experimental setup to resolve the functional cycle of Hsp70 in unprecedented spatial and temporal resolution. The method combines high-resolution NMR spectroscopy with embedded kinetic measurements to simultaneously resolve kinetic rates and structural information of the individual states of an Hsp70 functional cycle. We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP. Precision measurements connect the ATP hydrolysis rate (kcat) and the ADP lifetime (koff) to conventional bulk experiments and thus reveal that ADP-Pi release and not ATP hydrolysis is the limiting step of the cycle. Unlike commonly thought, the phosphate generated from ATP hydrolysis locks the ADP-Pi into the NBD, and thus decouples the ADP release rate from the effect of external factors such as the bulk phosphate and calcium concentration. The method will serve as a platform for studies of the Hsp70 protein family and their co-chaperones, including full-length constructs that have key roles in biogenesis and disease.

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Section: Single Turn-over Assaymentioning

confidence: 99%

Section: Combined Measurement Of the Functional Cycle Kinetic Parametersmentioning

confidence: 99%

Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain

Hiller,

Mas

2024

Preprint

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“…The binding effect between the charged molecule and the stationary phase is reversible. When changing the pH or eluting with a buffer solution, which gradually increases the ion strength, the substance bound by the ion exchange agent can exchange with the ions in the eluent and be eluted into the solution [59]. Due to differences in the charges of different proteins, their binding abilities to ion exchangers also vary, resulting in different orders of elution into the solution [60].…”

Section: Ion Exchange Chromatographymentioning

confidence: 99%

Recent Advances Regarding Polyphenol Oxidase in Camellia sinensis: Extraction, Purification, Characterization, and Application

Zou,

Zhang,

et al. 2024

Foods

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Polyphenol oxidase (PPO) is an important metalloenzyme in the tea plant (Camellia sinensis). However, there has recently been a lack of comprehensive reviews on Camellia sinensis PPO. In this study, the methods for extracting PPO from Camellia sinensis, including acetone extraction, buffer extraction, and surfactant extraction, are compared in detail. The main purification methods for Camellia sinensis PPO, such as ammonium sulfate precipitation, three-phase partitioning, dialysis, ultrafiltration, ion exchange chromatography, gel filtration chromatography, and affinity chromatography, are summarized. PPOs from different sources of tea plants are characterized and systematically compared in terms of optimal pH, optimal temperature, molecular weight, substrate specificity, and activators and inhibitors. In addition, the applications of PPO in tea processing and the in vitro synthesis of theaflavins are outlined. In this review, detailed research regarding the extraction, purification, properties, and application of Camellia sinensis PPO is summarized to provide a reference for further research on PPO.

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