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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.
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PMID:Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane. 171 97

One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.
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PMID:Immunochemical characterization of two isoforms of rat liver ecto-ATPase that show an immunological and structural identity with a glycoprotein cell-adhesion molecule with Mr 105,000. 183 73

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.
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PMID:Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes. 214 51

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.
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PMID:Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin. 215 Jul 94

The effect of myosin LC2 modifications (phosphorylation or selective proteolytic removal of a seven-residue N-terminal peptide) and partial or complete removal of the whole LC2 was studied under various conditions. (1) Actin binding in the absence of ATP is not influenced by the nature of the myosin species (phosphorylated, dephosphorylated or devoid of LC2). (2) A 50% inhibition of K+/EDTA-ATPase was obtained with actin concentrations hardly different when phosphorylated and dephosphorylated myosins were compared (of the order of 5 microM), whereas both myosin devoid of LC2 and myosin in which the LC2 N-terminal peptide has been removed required significantly higher concentrations of actin (13.0 +/- 2 and 12.0 +/- 2.0 microM, respectively). (3) Dissociation of the actomyosin complex at high ionic strength with nucleotides is not influenced by phosphorylation. (4) Actin activation of Mg2+-ATPase is enhanced when LC2 is phosphorylated; no activation enhancement is observed with myosin devoid of LC2. (5) Translational diffusion coefficient measurements of myosin in high-ionic-strength solutions indicate a tendency for LC2-deprived myosin to form autoassociation oligomers. It thus appears that a structural modification (partial cleavage or removal of LC2) induces important structural changes in myosin, pointing to a role for LC2 in the intrinsic conformation of the molecule and its interaction potentialities. Effects of LC2 removal at high ionic strength are best explained by interactions bearing no relationship to physiological functions. A physiologically significant effect of LC2 phosphorylation requires a minimum degree of organization (actomyosin complex) to be expressed in which LC2 could play the role of a return-spring in the cross-bridge mechanism.
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PMID:Influence of the regulatory light chain of fast skeletal muscle myosin on its interaction with actin in the presence and absence of ATP. 293 62

The rate of tryptic digestion of cardiac troponin C (cTNC) has been shown to be dependent on Ca2+ as was noted earlier for skeletal TNC (sTNC). Two representative peptides have been characterized on the basis of amino acid composition and partial amino terminal sequence analysis. Circular dichroism and fluorescence studies monitored their response to the presence of Ca2+. Their ability to form complexes with the ATPase inhibitory subunit of cardiac troponin (cTNI) was determined by urea - polyacrylamide gel electrophoresis and fluorescence experiments. The ability of these peptides to substitute for whole cTNC in restoring the ATPase activity of a partially inhibited synthetic actomyosin system was also explored. The N-terminal peptide 1-88 already contains a large amount of ordered structure, which indicates that the alpha-helices flanking binding site II of cTNC exist independently of Ca2+. Consequently this peptide shows limited increase in structure in the presence of Ca2+. It binds to cTNI independently of the presence of Ca2+ and could substitute for whole cTNC by relaxing the inhibitory effect of cTNI. The C-terminal peptide 103-158 has a low amount of secondary structure in the absence of Ca2+ but this increases dramatically in the presence of this cation. This peptide could only form a stable complex with cTNI in the presence of Ca2+ and was unable to release the inhibitory effect of cTNI.
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PMID:Trypsin digestion of bovine cardiac troponin C in the presence and absence of calcium. 293 34

When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain. The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions. The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates. This behaviour of the light chains-peptide complex mimics that of the intact molecule. The effect of bound light chains and bound actin on the susceptibility to tryptic digestion was studied using scallop S1(+LC) and S1(-LC) (EDTA S1 according to previous nomenclature). The heavy chains of both types of S1 are labile and have two main sites susceptible to proteolysis. Tryptic digestion on site A produces an N-terminal peptide of around 70 000 and a C-terminal 24 000 fragment from S1(+LC) and a 20 000 C-terminal fragment from S1(-LC); the latter is prone to further proteolysis. Thus S1(-LC), produced in the absence of bound regulatory light chain is shorter on the C-terminal end. Proteolysis on site A abolishes actin-activated ATPase activity; the latter is prevented by digesting acto-S1. The rate of tryptic digestion on site B is somewhat slower than on site A; when either S1 is split at this site an N-terminal 63 000 peptide is produced. The corresponding C-terminal peptide can be obtained from acto-S1 when hydrolysis on site A is prevented; this is estimated as around 31 000 derived from S1(+LC) and 28 000 derived from S1(-LC). The results are compared with similar experiments where vertebrate subfragments were digested by trypsin and the possible localization of the light-chain binding peptide in the intact heavy chain is discussed.
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PMID:Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide. 623 96

The presence of a formyl blocking group at the N-terminus of the ATPase inhibitor has been identified and the partial sequence of the N-terminal peptide has been determined by fast atom bombardment and field desorption coupled to mass spectrometry. Minor discrepancies in amino acid sequence of the inhibitor between the present and published data [(1981) Proc. Natl. Acad. Sci. USA 78, 7403-7407] are reported and its relationships with other inhibitors are briefly discussed.
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PMID:Mass spectral identification of the blocked N-terminal tryptic peptide of the ATPase inhibitor from beef heart mitochondria. 623 2

The protease subtilisin has been reported to cleave skeletal muscle G-actin between Met 47 and Gly 48 generating a core fragment of 33 kDa and a small N-terminal peptide, which remains attached to the core fragment [Schwyter, D. Phillips, M., & Reisler, E. (1989) Biochemistry 28, 5889-5895]. However, amino acid sequencing and mass spectroscopy of subtilisin cleaved-actin revealed two cleavage sites, one between Met 47 and Gly 48 and a second between Gly 42 and Val 43, generating an actin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we describe a procedure for purifying the actin core fragment and the attached N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromatography. After removal of the pentapeptide, the salt-induced polymerization of actin was abolished. However, the purified fragments could be polymerized by addition of salt plus myosin subfragment 1 or salt plus phalloidin as shown by sedimentation and fluorescence increase using N-(1-pyrenyl)iodoacetamide labeled actin. These results confirm earlier reports proposing that cleavage in the DNase I binding loop is affecting the ion induced polymerization of actin [Higashi-Fujime, S., et al. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subactin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct binding of subactin to DNase I was verified by gel filtration and to myosin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.
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PMID:Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop. 757 93

To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.
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PMID:Intersubunit and intrasubunit contact regions of Na+/K(+)-ATPase revealed by controlled proteolysis and chemical cross-linking. 759 71


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