Both native conformers of rabbit muscle adenylate kinase are active

Abstract

There are two forms of rabbit muscle adenylate kinase (AK) with different 8-anilino-1-naphthalenesulfonic acid (ANS) binding properties in equilibrium solution. One form (about 70%, denoted N 1 ) binds rapidly with ANS, whereas the other (about 30%, denoted N 2 ) does not. Furthermore, native forms of AK should adopt different conformations for binding with substrates and products, which should be pre-existing for performing its catalytic function. The present experiments demonstrate both forms of AK distinguished by ANS probe are active. The activity of N 2 is about 0.8 fold higher than N 1 and shows higher susceptibility to proteolysis by trypsin. This means that the native state of AK might be an ensemble of kinetically attainable conformers and the energy landscapes of AK folding should be rugged with more than one local minimum. ß

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

“…CsAK3 activity was measured according to the method described by Li and Pan (2000) and Tomasselli et al (1979). The formation of ADP was measured by a coupled assay using pyruvate kinase and lactate dehydrogenase.…”

Conjugated Polyelectrolyte Based Real-Time Fluorescence Assay for Adenylate Kinase

“…An analytical detection limit of ∼42 nM ADK is obtained from the linear range of the plot in Figure . This sensitivity is far below the concentration range of ADK which is generally assayed by commonly used coupled-enzyme assays in previous applications. ,,− …”

“…Several ADK assays have been developed based on chromatographic separation, pH-stat titration, bioluminescence, − and coupled-enzyme methods. , The chromatographic separation-based assay offers direct measurement of changes in the amount of the adenine nucleotides; however, it is laborious and time-consuming . The pH-stat assay suffers from low sensitivity and the inherent disadvantage that large quantities of enzyme and substrate are required .…”

“…For example, the assay to detect the forward reaction activity of ADK (Scheme ) is coupled with pyruvate kinase and lactate dehydrogenase and is carried out by monitoring the absorbance change of NADH at 340 nm concomitant with its oxidation. In a similar way, the assay to detect the reverse activity of ADK is coupled with hexokinase and glucose-6-phosphate dehydrogenase and carried out by monitoring the absorbance change of NADPH at the same wavelength accompanying the reduction of NADP . The colorimetric enzyme-coupled assays offer simple operation and potentially real-time detection; however, they are less sensitive compared with fluorometric assays, and their accuracy depends on other system factors such as the activity of coupled enzymes.…”

Conformational and functional significance of residue proline 17 in chicken muscle adenylate kinase

“…It was suggested that the conformational changes in AK be involved in proline isomerization [14,17,18]. In this work, proline 17 of chicken muscle AK has been replaced by glycine or valine using site‐specific mutagenesis to investigate the structure and functional role of the proline residue.…”