Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Cys127-Cys150 disulfide-bonded loop (L1) of the Torpedo californica Na,K-ATPase beta 1 subunit was substituted with the corresponding loop of the rat beta 1, mouse beta 2, or pig H,K-ATPase beta subunit. All the substituted mutant beta subunits assembled with the Na,K-ATPase alpha subunit in a trypsin-resistant manner. The mutants with L1 from the Na,K-ATPase beta subunit isoforms (rat beta 1 and mouse beta 2) each formed a functional complex with the Na,K-ATPase alpha subunit. On the other hand, the complex of the alpha subunit with the mutant beta subunit that was substituted with the pig H,K-ATPase beta subunit L1 was inactive as to ATP hydrolysis. Ser131 and Phe148 located within L1 of the pig H,K-ATPase beta subunit-substituted mutant were back-mutated to Pro131 and Arg148, respectively. The Phe148 to Arg mutation restored the ability of the mutant beta subunit substituted with the H,K-ATPase beta subunit L1 to form a functional complex with the alpha subunit. These results suggested that the Cys127-Cys150 loop of the Na,K-ATPase beta 1 subunit, especially Arg148, plays a critical role in the functional expression of Na,K-ATPase.
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PMID:Functional consequences of substitution of the disulfide-bonded segment, Cys127-Cys150, located in the extracellular domain of the Na,K-ATPase beta subunit: Arg148 is essential for the functional expression of Na,K-ATPase. 762 27

There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat alpha 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase alpha 1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the 32P was found on Ser-11, a site that is well conserved in Na,K-ATPase alpha 1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase alpha subunit sequences. This accounts for the observation that dog and pig alpha 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat alpha 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.
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PMID:Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C. 777 68

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.
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PMID:Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site. 812 8

The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.
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PMID:Substitutions of glutamate 781 in the Na,K-ATPase alpha subunit demonstrate reduced cation selectivity and an increased affinity for ATP. 857