scholarly journals Tracking the ATP-binding response in adenylate kinase in real time

2021 ◽  
Vol 7 (47) ◽  
Author(s):  
Fredrik Orädd ◽  
Harsha Ravishankar ◽  
Jack Goodman ◽  
Per Rogne ◽  
Lars Backman ◽  
...  
Keyword(s):  
Real Time ◽  
Adenylate Kinase ◽  
Atp Binding

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

2008 ◽  
Vol 412 (2) ◽  
pp. 315-321 ◽  
Author(s):  
Mohabir Ramjeesingh ◽  
Francisca Ugwu ◽  
Fiona L. L. Stratford ◽  
Ling-Jun Huan ◽  
Canhui Li ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Mechanism Of Action ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Residual Activity ◽  
Atp Binding ◽  
Cftr Protein ◽  
The Individual ◽  
Atp Binding And Hydrolysis

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain–domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

scholarly journals Real-Time Reverse Transcription-PCR Expression Profiling of the Complete Human ATP-Binding Cassette Transporter Superfamily in Various Tissues

2003 ◽  
Vol 49 (2) ◽  
pp. 230-238 ◽  
Author(s):  
Thomas Langmann ◽  
Richard Mauerer ◽  
Alexandra Zahn ◽  
Christoph Moehle ◽  
Mario Probst ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Diagnosis ◽  
Abc Transporter ◽  
Abc Transporters ◽  
Reverse Transcription ◽  
Complete Analysis ◽  
Atp Binding ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Atp Binding Cassette

Abstract Background: ATP-binding cassette (ABC) transporters are involved in many physiologic processes, such as lipid transport, sterol homeostasis, immune mechanisms, and drug transport, and cause various human inherited diseases. Thus, the analysis of ABC transporter mRNA expression profiles for basic research, especially in the field of lipid metabolism, for clinical diagnosis, and for monitoring of drug effects is of great interest. Methods: We have developed a rapid, accurate, and highly sensitive real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of all 47 currently known members of the ABC transporter superfamily. Our expression analysis is based on relative quantification using a calibration curve method. With our assay, expression monitoring of a large number of RNA samples in a 384-well format with only 50 ng of total RNA is possible. Results: In contrast to previous expression analyses of single ABC genes, our method allows the rapid and complete analysis of all ABC transporters in given RNA samples. We used our newly established expression panel to study the gene expression of all human ABC transporters in 20 different human tissues. As a result, we identified tissues with high transcriptional activity for ABC transporters. These organs are mainly involved in secretory function (adrenal gland), metabolic function (liver), barrier function (lung, trachea, small intestine), and tropic function (placenta, uterus). Conclusions: Our RT-PCR assay allows rapid, high-throughput transcriptional profiling of the complete ABC transporter superfamily and thus provides a new enabling tool for research, clinical diagnosis of disease, and drug testing and development.

Solution structure of the 45-residue magnesium ATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

Biochemistry ◽  
1988 ◽  
Vol 27 (10) ◽  
pp. 3588-3598 ◽  
Author(s):  
David C. Fry ◽  
D. Michael Byler ◽  
Heino Susi ◽  
Eleanor M. Brown ◽  
Stephen A. Kuby ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Solution Structure ◽  
Cd Spectroscopy ◽  
Atp Binding ◽  
Binding Peptide

scholarly journals ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

2013 ◽  
Vol 288 (38) ◽  
pp. 27692-27701 ◽  
Author(s):  
Christoph O. Randak ◽  
Qian Dong ◽  
Amanda R. Ver Heul ◽  
Adrian H. Elcock ◽  
Michael J. Welsh
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Atp Binding Site ◽  
Adenylate Kinases ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.

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