Identification of a cross‐linked double‐peptide from the catalytic site of the Ca2+‐ATPase of sarcoplasmic reticulum formed by the Ca2+‐ and pH‐dependent reaction with ATP γP‐imidazolidate

Gutowski-Eckel, Z; Mann, Karlheinz; Bäumert, Hans G.

doi:10.1016/0014-5793(93)80142-h

Cited by 10 publications

(5 citation statements)

References 21 publications

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“…The first one contains Thr-532 and Thr-533, which have been labeled by 8Na-ADP(Lacap£reetal., 1993). Thesecondone overlaps with a larger peptide recently cross-linked with the phosphorylation site (Gutowski-Eckel et al, 1993), and an analogous region of this peptide has also been labeled by 2N3-ATP within H+-ATPase (Davis et al, 1990). Thus these two regions are probably close to the adenosine part of the nucleotide but also not far from the phosphorylation site since the distance between the phosphate and the nitrene group of ANPP is close to 5 A (see Figure 7, top).…”

Section: Discussionmentioning

confidence: 85%

“…The general scheme is taken from MacLennan et al (1985). Asp-351 is phosphorylated by both ATP and Pj (Allen & Green, 1976) and is also cross-linked to residues Ile-625 and Met-700 (Gutowski-Eckel et al, 1993). Cys-471 is modified with the disulfide of the biotinyl-ITP (Kisonetal., 1989).…”

Section: Discussionmentioning

confidence: 99%

“…terminal phosphate group of ATP, since ATP molecules modified on the terminal phosphate, such as imidazolidate or carbodiimide ATP (Billetal., 1988;Murphy, 1990), inactivate the Ca-ATPase by forming an intramolecular cross-link between the Al and B fragments (Murphy, 1990;Gutowski-Eckel et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

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Interaction of 4-Azido-2-Nitrophenyl Phosphate, a Pseudosubstrate, with the Sarcoplasmic Reticulum Ca-ATPase

Lacapère¹,

Garin²

1994

Biochemistry

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In the dark, 4-azido-2-nitrophenyl phosphate (ANPP) is a phosphate analog which behaves like a simple energy-rich phosphate donor for the sarcoplasmic reticulum (SR) Ca-ATPase. Like p-nitrophenyl phosphate (pNPP), ANPP is hydrolyzed by the enzyme only in the presence of calcium and magnesium (K0.5 and Vmax are 0.3 mM and 60 nmol mg-1 min-1, respectively). After photoirradiation in the absence of magnesium, SR Ca-ATPase is specifically labeled by [32P]ANPP in the presence and in the absence of calcium. The presence of nucleotide in the medium provides some protection against photolabeling but less than phosphorylation by inorganic phosphate. The maximal stoichiometry of covalently bound ANPP extrapolates to 0.8 mol/mol of Ca-ATPase in the absence of magnesium. Autoradiography of a sodium dodecyl sulfate-polyacrylamide gel, after controlled trypsin digestion of the photolabeled protein, reveals that the radioactivity is incorporated in the B subfragment. Three radioactive polypeptides with approximate molecular masses of 55, 25, and 6 kDa are obtained depending on the digestion conditions. N-Terminal sequence analysis of the 50- and 25-kDa peptides reveals the same sequence beginning at Ala-506, whereas two different sequence beginning at Ala-506 and Phe-584 are observed in the 6-kDa peptides.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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Interaction of 4-Azido-2-Nitrophenyl Phosphate, a Pseudosubstrate, with the Sarcoplasmic Reticulum Ca-ATPase

Lacapère¹,

Garin²

1994

Biochemistry

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“…The 56 kDa that exhibited by enterobacteria and from the inhibition of GS band could represent a different cross-linked product between by metabolites described in Gram-positive bacteria [20]: evithe 14 kDa protein and the GS subunit that gave a different dently, the system chosen by Synechocystis to regulate GS acelectrophoretic mobility, as occur in one of the subunit of tivity requires a constitutive expression of the inactivating proCa2+-ATPase of sarcoplasmic reticulum due to an intramolecurein in order to be available when the nitrogen source changes; lar cross-linking in this subunit [17].…”

Section: 2 Cross Linked Complex Characterizationmentioning

confidence: 99%

A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein

Reyes

Florencio

1995

FEBS Letters

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s (in 15 s periods). The homogenated was centrifuged at 40,000 × g for Cyanobacteria; Ammonium assimilation; Enzyme inactivation 15 min and the resulting supernatant constituted the cell-free extract. Polyacrylamide gel electrophoresis and Western blot analysisSamples of crude extract were run in polyacrylamide SDS gel, using 6% (w/v) to 10% acrylamide gradient gel, according to Laemmli [8]. IntroductionMarker proteins were Prestained SDS-PAGE Standards (low and high range) from Bio-Rad Laboratories. Non-denaturing gel electrophoresis was carried out in 6.25% (w/v) acrylamide gels and run at 4°C. IdentiGlutamine synthetase (GS) (EC 6.3.1.2) is a key enzyme in fication of the glutamine synthetase activity in non-denaturing gel elec-2+ the nitrogen assimilation process in most microorganisms. This trophoresis was carried out by the Mn -dependent y-glutamyl-transcentral role of GS requires the synthesis and the activity of this ferase assay [9], modified as follows: after non-denaturing PAGE, the gel was incubated at 30°C in a reaction mixture containing, in a final enzyme to be finely regulated in response to the available nitrovolume of 10 ml, 600/zmol of HEPES-NaOH buffer (pH 7.0), 400/zmol gen source [1,2]. GS from Escherichia coli and other GramofL-glutamine,40/lmolofMnCl:, 600/zmolofhydroxylamine, 10/lmol negative bacteria is modulated at the activity level by a mechaof ADP and 200/~mol of sodium arsenate. After 20 rain the reaction nism of adenylylation/deadenylylation of the enzyme, being was stopped by transferring the gel to FeCI 3 in acid solution that stains active the deadenylylated form [3]. the bands where y-glutamylhydroxamate was formed by the GS activityIn cyanobacteria, ammonium assimilation takes place mainly [9]. Western blot analysis of crude extract samples subjected to denaturby the glutamine synthetase-glutamate synthase (GS-GOGAT) ing or native electrophoresis were carried out as previously described pathway [4]. In the unicellular cyanobacterium Synechocystis [10]. Purified polyclonal monospecific antibodies obtained against sp. PCC 6803 we have previously reported a short-term ammoSynechococcus sp. PCC 6301 glutamine synthetase were used [11].nium-promoted GS inactivation, in a process that comprises 2.4. Cross-linking treatment the non-covalent binding of a phosphorylated factor, whose Cross-linking reactions were performed at 25°C in 50 mM HEPESnature was then unknown [5,6]. The inactive GS could be reacNaOH buffer, pH 7.0, by addition of l-ethyl-3-(3-dimethylaminoprotivated in vitro by different treatments that include alkaline pyl)carbodiimide (EDC) to a final concentration of 4 mM. Final volphosphatase, increasing pH or high ionic strength [6]. In this.ume of cross-linking reaction was 200 ~tl with about 2 mg of total protein. Aliquots of 20/zl were taken at various intervals and mixed study we show for the first time, by using Western blot analysis with an equal volume of dissociation buffer (1% SDS, 1% 2-mercapand cross-linking techniques, that the ammonium-promoted toethanol, 30/...

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“…These data suggest that domains P and N are brought close together during ATP hydrolysis or in the occluded state stabilized by CrATP. Ca 2+ binding affects the availability of Lys492 for reaction with adenosine triphosphopyridoxal (Yamamoto, 1989), facilitates the cross-linking of Asp351 to Lys684 by ATP imidazolidate (Gutowski-Eckel et al, 1993), changes the reactivities of the enzyme with ATP and P i (Mintz & Guillain, 1997) and alters the sensitivity of cleavage sites to trypsin (Dux & Martonosi, 1983b;Imamura et al, 1984;Andersen & Jorgensen, 1985;Dux et al, 1985b), proteinase K (Yuul et al, 1995), V8 protease (Danko et al, 2001a;2001b) and vanadate (Vegh et al, 1990;Molnar et al, 1991;Hua et al, 2000). These changes are presumably brought about by a Ca 2+ -induced reorientation of the transmembrane helices that are transmitted to the cytoplasmic domains.…”

Section: Three Dimensional Structure Of Serca1a By X-ray Crystallogramentioning

confidence: 99%

The structure of the Ca2+-ATPase of sarcoplasmic reticulum.

Martonosi

Pikula²

2003

Acta Biochim Pol

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In this article the morphology of sarcoplasmic reticulum, classification of Ca(2+)-ATPase (SERCA) isoenzymes presented in this membrane system, as well as their topology will be reviewed. The focus is on the structure and interactions of Ca(2+)-ATPase determined by electron and X-ray crystallography, lamellar X-ray and neutron diffraction analysis of the profile structure of Ca(2+)-ATPase in sarcoplasmic reticulum multilayers. In addition, targeting of the Ca(2+)-ATPase to the sarcoplasmic reticulum is discussed.

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Identification of a cross‐linked double‐peptide from the catalytic site of the Ca²⁺‐ATPase of sarcoplasmic reticulum formed by the Ca²⁺‐ and pH‐dependent reaction with ATP γP‐imidazolidate

Cited by 10 publications

References 21 publications

Interaction of 4-Azido-2-Nitrophenyl Phosphate, a Pseudosubstrate, with the Sarcoplasmic Reticulum Ca-ATPase

Interaction of 4-Azido-2-Nitrophenyl Phosphate, a Pseudosubstrate, with the Sarcoplasmic Reticulum Ca-ATPase

A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein

The structure of the Ca2+-ATPase of sarcoplasmic reticulum.

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