QuantiChrom EnzyChrom Kit

The column, fraction collector, and autosampler were set to 4 °C. A volume of 15 μl of 2C protein (75 μg) was injected at a flow rate of 0.5 ml min−1 with a chromatogram run time of ∼25 minutes. Data was analyzed using the ASTRA QuantiChrom - EnzyChrom products software from Wyatt. As a single peak with a molar mass corresponding to a dimer of 2C protein was observed by SEC-MALS, BioSAXS data were subsequently collected directly with no further purification via SEC-MALS fractions.
SsRNA 3 and  ssRNA 5 were 32P-labeled, indicated by a star. RNA unwinding by 2C was assessed by phosphorimaging of native polyacrylamide gels. HCV NS3 (0.5 μM) or WT 2C (2 μM) was incubated with 10 nM Duplex 1 or Duplex 2 using two different reaction conditions . The unwinding reactions were quenched after 2, 5, and 15 minutes.

The volume of enzyme added to any reaction was always less than or equal to one-tenth the total reaction volume. Rates are defined as μM ADP formed min−1 μM 2C−1. When defining the rate, timepoints were taken during the linear portion of the reaction with less than 20% product formed. Inclusion of 2C in this family makes it likely that 2C would fulfill some type of helicase function, but neither widespread demonstration nor a specific biological target of helicase activity has yet been identified. Similarly, the helicase and/or translocase activity might facilitate encapsidation of viral RNA during morphogenesis. Again, the biological, biochemical, or biophysical evidence to support these assumptions is sparse.
6F to L), suggesting a role for the amino terminus in formation and/or stability of the 2C2 dimer. Both Duplex substrates contain a 9-bp region; sequence is highlighted. Duplex 1 is flanked by 20-nt single-stranded tails on the 5′ and 3′-ends of ssRNA 2. Duplex 2 is forked, such that there are two single-stranded tails on either side of the duplex, formed by ssRNA 4 and ssRNA 5.

Gels were visualized by phosphorimaging. Where kmax is the maximum rate with a substrate concentration of 0.5 mM ATP, C is a constant, and kobs the observed rate (μM ADP min−1 μM 2C−1). Assays can be directly performed on raw biological samples i.e., in the presence of lipid, protein and minerals such as magnesium, iron and zinc. All data are incorporated into the article and its online supplementary material. Constructs and full data sets presented in this study are available upon request to the corresponding author.
Please check for further notifications by email. We are optimistic that the groundwork established here will facilitate elucidation of high-resolution structures of the catalytically competent state. AThe apparent dissociation constant is presented ± standard error with 95% confidence interval provided in brackets below. The procedure involves addition of a single working reagent and incubation for 3 min. Can be readily automated as a high-throughput assay for thousands of samples per day. Your order for QuantiChrom products is prepared and secured by our specialists.
RNA-stimulated ATPase activity observed for members of enteroviral species A–D. Purified NΔ39 2C proteins (5 μg each) from Coxsackievirus B3 , Enterovirus A71 (EV-A71), and Enterovirus D68 (EV-D68) were visualized on a 15% polyacrylamide gel and was stained with Coomassie. RNA-stimulated enteroviral 2C ATPase activity.

Binding of RNA and ATP to 2C occurs using a two-step process, which suggests specific interactions between 2C, 2′-hydroxyls of RNA, and both the base and ribose of ATP. These key principles revealed by studying PV 2C protein extend to EV-A71, CVB3, and EV-D68. Is there a biological role for a dimeric, RNA-dependent ATPase during the enteroviral lifecycle? Further studies will be required to address this question. If not, perhaps other scaffolds for may exist to assemble hexamers during genome replication.

The goal of this study was to establish a framework to guide future studies of the specificity, kinetic and chemical mechanisms, and inhibition of the enteroviral 2C ATPase using PV 2C as our model system. This observation is consistent with RNA binding measured by polarization actually reflecting formation of the 2C dimer. Fold stimulation is the quotient of linear rate of RNA-stimulated activity divided by RNA-independent activity. What these experiments do not reveal is whether the dissociation constant measured reflects a value for the 2C dimer, RNA-bound 2C dimer, or some combination thereof. Below, we perform experiments to distinguish between these possibilities. Where X is the concentration of protein, Y is degree of polarization, Kd,app is the apparent dissociation constant, and Ymax is the maximum value of Y.
Our studies make a compelling case for the existence of a conformation of the PV 2C-ATP complex that links binding of the correct nucleotide to catalytic competence of the active site. As a first step towards identification of other determinants of ATP interrogated in the second step of binding that are required for catalysis, we have investigated the activity of several analogues of ATP. Together, these data suggest that a single binding site exists for binding to single-stranded nucleic acid, and that the primary determinant for binding to this site is the phosphodiester backbone. Use of high protein concentrations is a requirement for this experiment, so it is not possible to glean any insight into the equilibrium dissociation constant for the dimer from this experiment.

However, the inability to observe hexamers over this range of concentration suggests that the equilibrium dissociation constant for such structures must be quite high. BioAssay Systems calcium assay kit is designed to measure calcium directly in biological samples without any pretreatment. A phenolsulphonephthalein dye in the kit forms a very stable blue colored complex specifically with free calcium. The intensity of the color, measured at 612 nm, is directly proportional to the calcium concentration in the sample.