Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites.

Capasso, Juan M.; Hoving, Sjouke; Tal, Daniel M.; Goldshleger, Rivka; Karlish, S. J. D.

doi:10.1016/s0021-9258(18)48408-6

Cited by 153 publications

(84 citation statements)

References 37 publications

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“…This is somehow unexpected since the cleavage of other P-ATPases in this region only takes place after extensive proteolytic digestion [34,51]. Indeed, the trypsin cut of the Na + /K + ATPase at Gln737 near the amino terminal end of the cytosolic portion of M5 helix occurs only after a long exposure to the protease and generates the so-called protease-resistant K + -occluding 19 kDa membranes [34]. Noteworthy, the stabilization of the 19 kDa Na + /K + ATPase membranes requires the presence of the transported ion (K + ) while in its absence a proteolytic fragment containing M5-M6 hairpin is spontaneously released from the membrane [51].…”

Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 98%

“…Limited proteolysis is a well characterized strategy to gain information on the structure of proteins at a relatively low resolution and has been extensively used to advance the knowledge of different P-ATPases [29][30][31][32][33][34][35][36][37][38][39][40][41][42]. While the cleavage sites of each protease have specific primary sequence constrains, the precise location where the cuts occur are mostly determined by the segmental exposure and flexibility [27,28].…”

Section: The Structure Of Spf1p Detected By Limited Proteolysismentioning

confidence: 99%

“…The structure of several P-ATPases have been thoroughly examined by limited proteolysis, in particular that of the Na + /K + ATPase, SERCA and those like PMCA or Drs2p which have flexible terminal regulatory domains [29][30][31][32][33][34][35][36][37][38][39][40][41][42]. These studies show that despite the differences in primary sequence, the preferential proteolytic attack occur in well-defined regions, as it would be expected from sharing a similar structure and domain organization [48,49].…”

Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 99%

“…In contrast, our results suggest that Spf1p is cleaved by chymotrypsin primarily in the M4-M5 loop C-terminal portion corresponding to the P-domain near M5. This is somehow unexpected since the cleavage of other P-ATPases in this region only takes place after extensive proteolytic digestion [34,51]. Indeed, the trypsin cut of the Na + /K + ATPase at Gln737 near the amino terminal end of the cytosolic portion of M5 helix occurs only after a long exposure to the protease and generates the so-called protease-resistant K + -occluding 19 kDa membranes [34].…”

Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 99%

“…In general, the actual cleavage sites of a native protein are determined more by the tertiary structure than by the specificity of the protease [28]. Limited proteolysis of P-ATPases has been a particularly useful technique for the study of the topology, domain function and conformational changes of P-ATPases [29][30][31][32][33][34][35][36][37][38], and has revealed the presence of non-essential regulatory domains in several members of the family, i.e. the auto-inhibitory domains of the plasma membrane Ca 2+ pumps (PMCA), the yeast plasma membrane H + -ATPase (Pma1p) and the lipid flippase Drs2p [39][40][41][42].…”

Section: Introductionmentioning

confidence: 99%

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Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

et al. 2021

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The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.

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Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 98%

Section: The Structure Of Spf1p Detected By Limited Proteolysismentioning

confidence: 99%

Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 99%

Section: Comparison Of the Limited Proteolysis Pattern Of Spf1p With That Of Other P-atpasesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

et al. 2021

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Digitalisforschung in Berlin‐Buch – Rückblick und Ausblick

et al. 1995

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Der Arzt William Withering führte 1785 mit seinem Buch „An Account of the Foxglove and some of its Medical Uses”︁ Präparate aus Digitalisblättern in die Therapie der Wassersucht (des Herzversagens) ein und erklärte: „The following remarks consist partially of matter of fact, and partially of opinion. The former will be permanent; the latter must vary with the detection of error, or the improvement of knowledge. I hazard them with diffidence, and hope they will be examined with candour.”︁ Diese Bemerkungen sind hier vom Seniorautor angeführt, da er die Schwierigkeit sieht, einen ausgewogenen Bericht über sein lebenslanges Forschungsprojekt zur Digitalis‐Weiterentwicklung zu bieten. Seine Entscheidung, sich der Digitalisforschung zu widmen, entstand am Krankenbett, an dem er als Arzt die furchtbaren Endstadien des Herzversagens erlebte, in denen für die Kranken keine wirkliche Hilfe mehr möglich ist. Unglücklicherweise paßte dieses Vorhaben nicht in das vom Wissenschaftsministerium der DDR dekretierte Forschungsprogramm, so daß er das Digitalisprojekt zugunsten von Biomembran‐Untersuchungen einstellen sollte. Glücklicherweise entging er dem Verdikt durch Etikettierung der digitalisartig wirkenden Steroide als chemische Sonden für die Zellmembran‐lokalisierte Na+/K+ ‐transportierende ATPase, die er gerade als den Digitalisrezeptor erkannt hatte. Über die Arbeit der Autoren wird hier erstmals im Zusammenhang berichtet. Ziel des Überblicks ist, die Forschung für die Lösung einer großen Aufgabe zu fördern: die Entwicklung von Steroidwirkstoffen zur Verhinderung und Heilung der Herzinsuffizienz.

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Digitalis Research in Berlin–Buch—Retrospective and Perspective Views

Repke¹,

Megges²,

Weiland³

et al. 1995

Angew. Chem. Int. Ed. Engl.

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“The following remarks consist partially of matter of fact, and partially of opinion. The former will be permanent; the latter must vary with the detection of error, or the improvement of knowledge. I hazard them with diffidence, and hope they will be examined with candour.” These declarations, which stem from the famous book “An Account of the Foxglove and some of its Medical Uses” by physician William Withering in 1785 in which he introduced preparations from digitalis leaves in the therapy of dropsy (cardiac failure), are cited here by the senior author because of his awareness of the difficulties in presenting a balanced report on his life‐long research project on the further development of digitalis. His decision to devote himself to digitalis research originated at the bedside, when as a physician he experienced the grim final stages of cardiac failure in which no real help for the patients is possible. Unfortunately, his research project did not fit into the research program decreed by the Ministry of Science of the German Democratic Republic, so that he was ordered to stop the digitalis project in favor of biomembrane studies. Fortunately, he got round the ban simply by labeling the digitalis‐like acting steroids as probes for the cell membrane‐located Na+/K+‐transporting ATPase which he had just recognized as the digitalis target (receptor) enzyme. These and other ventures by the authors are collated here for the first time. The aim of this review is to foster straightforward research for solving a major challenge: the development of steroidal drugs for the prevention and cure of cardiac failure.

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Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites.

Cited by 153 publications

References 37 publications

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

Digitalisforschung in Berlin‐Buch – Rückblick und Ausblick

Digitalis Research in Berlin–Buch—Retrospective and Perspective Views

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