Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities

Abstract

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.

Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs

“…Here we confirmed the substrate inhibition of Dh-mPPase ( Figure 3 ) by using a refined assay protocol. Specifically, we considered the PP i interference with P i assay ( Figure S1 ) and avoided the use of tetramethylammonium hydroxide in the buffer preparation after noting that some batches of this alkali supplied in glass containers have an adverse effect on PPase activity [ 28 ]. It was also confirmed that the substrate, Mg 2 PP i , retains solubility up to 1.2 mM during the activity assay at 25 °C [ 28 ].…”

“…A note on the activity assay is appropriate because its accuracy/precision was essential for deriving reliable reaction schemes containing multiple enzyme species. Because the assay was continuous and sensitive, it allowed a reliable estimation of initial velocity at a submicromolar Mg 2 PP i concentration [ 28 ], as strictly required by the low K m1 value. The lowest total PP i concentration (corresponding to 0.5 µM Mg 2 PP i at 5 mM Mg 2+ ) was 0.79 µM, which yielded 1.58 µM P i and produced a 7 cm signal on recorder paper upon complete hydrolysis.…”

“…The activity assay medium (typically 25 mL in volume) contained 0.1 M MOPS-KOH buffer, pH 7.2, and varied concentrations of free Mg 2+ ion (added as MgCl 2 ), Mg 2 PP i complex, and other indicated additions. Reactions were initiated by adding IMV suspension (0.01–0.24 mg protein), and P i accumulation due to PP i hydrolysis was continuously recorded for 2–3 min at 25 °C using an automated P i analyzer [ 28 ] at a sensitivity of 4–20 µM P i per recorder scale. The IMV amount was varied to achieve similar measured rates at different substrate, inhibitor, or Mg 2+ concentrations and, hence, the equal relative error of measurement for each data point [ 28 ].…”

“…Reactions were initiated by adding IMV suspension (0.01–0.24 mg protein), and P i accumulation due to PP i hydrolysis was continuously recorded for 2–3 min at 25 °C using an automated P i analyzer [ 28 ] at a sensitivity of 4–20 µM P i per recorder scale. The IMV amount was varied to achieve similar measured rates at different substrate, inhibitor, or Mg 2+ concentrations and, hence, the equal relative error of measurement for each data point [ 28 ]. The measured rate values were then normalized to the same IMV amount by calculating specific activities.…”

“…The total concentrations of MgCl 2 and PP i required to maintain the desired concentrations of Mg 2 PP i complex (presumed actual substrate [ 20 , 35 , 42 ]) and free Mg 2+ ion in the activity assay mixture at pH 7.2 were calculated using the apparent dissociation constants for the MgPP i and Mg 2 PP i complexes listed in Table 3 . These apparent constants were calculated from the stabilities of individual complexes formed between PP i , H + , Mg 2+ , K + , and Na + , as described before [ 28 ]. The same algorithm was used to calculate the apparent dissociation constants for MgHEDP and Mg 2 HEDP complexes at pH 7.2 in the presence of 50 mM K + from comparable data on the individual complexes formed by this PP i analog [ 43 ].…”