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The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase alpha-subunit is essential for K+ activation of the enzyme activity.

ABSTRACT: To investigate the role of Glu820, located in transmembrane domain M6 of the alpha-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820-->Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40-100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10-20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy) imidazo [1,2a]-pyridine (SCH28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the alpha-subunit.

SUBMITTER: Hermsen HP

PROVIDER: S-EPMC1219377 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Project description:Background and methodsMultiple Na+/K+-ATPase (NKA) α-subunit isoforms express differentially in response to salinity transfer in teleosts but we observed that the isoform nomenclature is inconsistent with the phylogenetic relationship of NKA α-genes. We cloned the catalytic NKA α-subunit isoforms in eels and medaka, analyzed the time course of their expressions in osmoregulatory tissues after transfer from freshwater (FW) to seawater (SW), and performed phylogenetic analyses to deduce an evolutionary scenario that illustrates how various duplication events have led to the current genomic arrangement of NKA α-genes in teleosts.Results and discussionFive and six α-subunits were cloned in eels and medaka respectively. In eels, the commonly-reported α1a and α1b isoforms were absent while the α1c isoform was diversified instead (α1c-1, α1c-2, α1c-3, α2, and α3 in eels). Phylogenetic estimation indicated that the α1a and α1b isoforms from salmon, tilapia, and medaka were generated by independent duplication events and thus they are paralogous isoforms. Re-examination of expression changes of known isoforms after salinity challenge revealed that the isoforms selected as predominant SW-types varied among teleost lineages. Diversification of α1 isoforms occurred by various types of gene duplication, or by alternative transcription among tandem genes to form chimeric transcripts, but there is no trend for more α1 copies in euryhaline species. Our data suggest that the isoform switching between FW (α1a predominates) and SW (α1b predominates) that occurs in salmonids is not universal in teleosts. Instead, in eels, α1c-1 was the major α-subunit upregulated gill, intestine, and kidney in SW. Localization of both NKA mRNA and protein showed consistent upregulation in gill and intestine in SW eels, but not in renal distal and collecting tubules, where low transcript expression levels were accompanied by high protein levels, suggesting a tissue-specific translational regulation that determines and fine-tunes the NKA expression. In medaka, α1b was upregulated in SW in anterior intestine while most other α-subunit isoforms were less responsive to salinity changes.ConclusionBy integrating gene expression and phylogenetic results, we propose that the major NKA α-subunits for SW acclimation were not ancestrally selected, but rather were flexibly determined in lineage-specific fashion in teleosts.

Project description:Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multistep proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ∼35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask examination of the kinetics of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [Re(I)] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET), and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively "tight" subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.

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The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase alpha-subunit is essential for K+ activation of the enzyme activity.

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