Nucleotide Binding and Nucleotide Hydrolysis Properties of the ABC Transporter MRP6 (<i>ABCC6</i>)
Bibliographic record
Abstract
Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".