PMID- 10569935
OWN - NLM
STAT- MEDLINE
DCOM- 19991216
LR  - 20191210
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 47
DP  - 1999 Nov 23
TI  - Overlapping effects of S3 stalk segment mutations on the affinity of Ca2+-ATPase 
      (SERCA) for thapsigargin and cyclopiazonic acid.
PG  - 15522-7
AB  - Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to 
      match (in the majority of cases) corresponding sequences of the 
      Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence 
      of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic 
      acid (CPA) were then determined. Extensive chimeric mutations on the large 
      cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments 
      produced little or no modification of the Ca(2+)-ATPase sensitivity to either 
      inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase 
      sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the 
      apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited 
      mutations within the same S3 segment, however, affected differently the 
      concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. 
      Specifically, single mutation of Phe256 to Val increased 20-fold the apparent 
      K(i) for TG, while having very little effect on the apparent K(i) for CPA. These 
      findings indicate significant overlap of the TG and CPA binding domains within 
      the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein 
      residue is dependent on the structures of the two inhibitors. Saturating 
      concentrations of either or both TG and CPA produce an identical reduction of the 
      affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at 
      any time due to significant overlap of their binding domains. It is suggested 
      that perturbations produced by binding of either inhibitor within the stalk 
      segment interfere with the long-range functional linkage between ATP utilization 
      in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.
FAU - Ma, H
AU  - Ma H
AD  - Departamento de Bioquimica y Biologia Molecular A, Veterinaria, Universidad de 
      Murcia, Spain.
FAU - Zhong, L
AU  - Zhong L
FAU - Inesi, G
AU  - Inesi G
FAU - Fortea, I
AU  - Fortea I
FAU - Soler, F
AU  - Soler F
FAU - Fernandez-Belda, F
AU  - Fernandez-Belda F
LA  - eng
GR  - P01 HL27867/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (2',3'(O)-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Indoles)
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 67526-95-8 (Thapsigargin)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 7.2.2.10 (Calcium-Transporting ATPases)
RN  - X9TLY4580Z (cyclopiazonic acid)
SB  - IM
MH  - Adenosine Triphosphate/analogs & derivatives/metabolism
MH  - Animals
MH  - COS Cells
MH  - Calcium-Transporting ATPases/*genetics/*metabolism
MH  - Chickens
MH  - Cytosol/drug effects/metabolism
MH  - DNA Mutational Analysis
MH  - Endoplasmic Reticulum/*enzymology
MH  - Enzyme Inhibitors/metabolism/pharmacology
MH  - Fluorescent Dyes/metabolism
MH  - Indoles/*metabolism/pharmacology
MH  - Mutagenesis, Site-Directed
MH  - Peptide Fragments/*genetics/metabolism
MH  - Protein Binding/genetics
MH  - Rabbits
MH  - Recombinant Fusion Proteins/chemistry/genetics/metabolism
MH  - Thapsigargin/*metabolism/pharmacology
EDAT- 1999/11/26 00:00
MHDA- 1999/11/26 00:01
CRDT- 1999/11/26 00:00
PHST- 1999/11/26 00:00 [pubmed]
PHST- 1999/11/26 00:01 [medline]
PHST- 1999/11/26 00:00 [entrez]
AID - bi991523q [pii]
AID - 10.1021/bi991523q [doi]
PST - ppublish
SO  - Biochemistry. 1999 Nov 23;38(47):15522-7. doi: 10.1021/bi991523q.