Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (αβ)2 dimer

Santos, Hérica de Lima; Ciancaglini, Pietro

doi:10.1016/s1096-4959(03)00139-8

Cited by 17 publications

(1 citation statement)

References 50 publications

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“…Other factors, such as interactions between the phospholipid DPPE and the DPPS used in the preparation of the PS-CPLX nucleator, cannot be completely ruled out. The inhibition of NKA by ouabain in proteoliposomes and in this assay condition has been extensively discussed by our group [ 62 , 63 , 84 , 85 , 86 , 87 ]. Nevertheless, the addition of ouabain did not completely prevent the ATP nucleation process, probably due to the occluded ouabain binding site, which turned to the lumen of proteoliposomes, but even the addition of detergent was not able to improve the inhibition and abrogate mineral formation.…”

Section: Resultsmentioning

confidence: 99%

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Sebinelli

Andrilli

Favarin

et al. 2022

IJMS

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Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg−1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69–72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8–13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3–8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm−1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3–8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

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Section: Resultsmentioning

confidence: 99%

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Sebinelli

Andrilli

Favarin

et al. 2022

IJMS

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Influence of enzyme conformational changes on catalytic activity investigated by circular dichroism spectroscopy

Rigos

Santos

Thedei

et al. 2003

Biochem Molecular Bio Educ

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Enzyme activity is dependent on native conformational integrity. Here we present a simple laboratory exercise based on dichroism spectroscopy in which the change in enzyme structure induced by denaturation is correlated with the loss of catalytic activity. The results of circular dichroism spectra show that enzyme denaturation by either trifluoroethanol (enhancement of ␣-helix structure) or guanidinium chloride (reduction of ␣-helix and enhancement of random coil structure) leads to a concomitant reduction in enzyme activity, which demonstrates the relationship between structure and catalytic activity of the enzyme. This simple experimental approach demonstrates that only a single native protein (enzyme) conformation has the ability to catalyze substrate hydrolysis.

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Identification, purification and partial characterization of low molecular weight protein inhibitor of Na+/K+-ATPase from pulmonary artery smooth muscle cells

et al. 2014

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We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α₂β₁ and α₁β₁ isozymes of Na(+)/K(+)-ATPase, where α₂ is more sensitive than α₁. The inhibitor interacted reversibly to the E1 site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.

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Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (αβ)2 dimer

Cited by 17 publications

References 50 publications

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Influence of enzyme conformational changes on catalytic activity investigated by circular dichroism spectroscopy

Identification, purification and partial characterization of low molecular weight protein inhibitor of Na+/K+-ATPase from pulmonary artery smooth muscle cells

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