Calcium in the myocardial cell is highly compartmentalized and a fast,
an intermediate, a slow and a nonexchangeable calcium pool have been
described. The fast pool contains 66\% of the total cell exchangeable
calcium in cultured neonatal rat heart cells with a t1/2 of less
than 1.5 sec. Though the cellular origin of this fast pool is unknown,
its rapidity and its displacement by La3+ most likely places it at
the sarcolemma or at least in rapid equilibrium with the sarcolemma.
We isolated the sarcolemma of cultured neonatal rat heart cells using
the gas-dissection technique, which yields a pure sarcolemmal preparation
in less than a second, thereby precluding membrane changes which
might occur during conventional plasma membrane isolation. We determined
the calcium binding characteristics of these membranes, using an
on-line technique to monitor 45Ca, which allows measurement of 45Ca
binding characteristics in the presence of unbound 45Ca. Two classes
of calcium binding sites were determined: (i) Kd of 13 microM, capacity
7 nmol/mg and (ii) Kd of 1.1 mM, capacity of 84 nmol/mg. To assess
the molecular origin of the sarcolemmal calcium binding we treated
the membranes with a variety of enzymes. Protease or neuraminidase
treatment did not cause large changes in these parameters. Simultaneous
treatment with two different phospholipases C or the extraction of
the lipids with isopropanol resulted in a dramatic loss of the low-affinity
binding sites. These results, in association with previously defined
sarcolemmal phospholipid distribution, places the low-affinity binding
sites at the cytoplasmic leaflet. The physiological implication of
this localization as it pertains to cellular calcium exchange is
discussed.
%0 Journal Article
%1 Post_1992_49
%A Post, J. A.
%A Langer, G. A.
%D 1992
%J J. Membr. Biol.
%K 1-Propanol, 1404340 Animals, Binding C, Calcium Calcium, Cell Cells, Compartmentation, Cultured, Dissection, Endopeptidases, Gov't, Lanthanum, Models, Myocardium, Neuraminidase, Newborn, Nifedipine, Nitrogen, Non-U.S. P.H.S., Phospholipase Phospholipids, Radioisotopes, Rats, Research Reticulu, Ryanodine, Sarcolemma, Sarcoplasmic Sites, Support, Terpenes, Thapsigargin, Theoretical, U.S. m,
%N 1
%P 49--57
%R 10.1007/BF00232054
%T Sarcolemmal calcium binding sites in heart: I. Molecular origin in
"gas-dissected" sarcolemma.
%U http://dx.doi.org/10.1007/BF00232054
%V 129
%X Calcium in the myocardial cell is highly compartmentalized and a fast,
an intermediate, a slow and a nonexchangeable calcium pool have been
described. The fast pool contains 66\% of the total cell exchangeable
calcium in cultured neonatal rat heart cells with a t1/2 of less
than 1.5 sec. Though the cellular origin of this fast pool is unknown,
its rapidity and its displacement by La3+ most likely places it at
the sarcolemma or at least in rapid equilibrium with the sarcolemma.
We isolated the sarcolemma of cultured neonatal rat heart cells using
the gas-dissection technique, which yields a pure sarcolemmal preparation
in less than a second, thereby precluding membrane changes which
might occur during conventional plasma membrane isolation. We determined
the calcium binding characteristics of these membranes, using an
on-line technique to monitor 45Ca, which allows measurement of 45Ca
binding characteristics in the presence of unbound 45Ca. Two classes
of calcium binding sites were determined: (i) Kd of 13 microM, capacity
7 nmol/mg and (ii) Kd of 1.1 mM, capacity of 84 nmol/mg. To assess
the molecular origin of the sarcolemmal calcium binding we treated
the membranes with a variety of enzymes. Protease or neuraminidase
treatment did not cause large changes in these parameters. Simultaneous
treatment with two different phospholipases C or the extraction of
the lipids with isopropanol resulted in a dramatic loss of the low-affinity
binding sites. These results, in association with previously defined
sarcolemmal phospholipid distribution, places the low-affinity binding
sites at the cytoplasmic leaflet. The physiological implication of
this localization as it pertains to cellular calcium exchange is
discussed.
@article{Post_1992_49,
abstract = {Calcium in the myocardial cell is highly compartmentalized and a fast,
an intermediate, a slow and a nonexchangeable calcium pool have been
described. The fast pool contains 66\% of the total cell exchangeable
calcium in cultured neonatal rat heart cells with a t1/2 of less
than 1.5 sec. Though the cellular origin of this fast pool is unknown,
its rapidity and its displacement by La3+ most likely places it at
the sarcolemma or at least in rapid equilibrium with the sarcolemma.
We isolated the sarcolemma of cultured neonatal rat heart cells using
the gas-dissection technique, which yields a pure sarcolemmal preparation
in less than a second, thereby precluding membrane changes which
might occur during conventional plasma membrane isolation. We determined
the calcium binding characteristics of these membranes, using an
on-line technique to monitor 45Ca, which allows measurement of 45Ca
binding characteristics in the presence of unbound 45Ca. Two classes
of calcium binding sites were determined: (i) Kd of 13 microM, capacity
7 nmol/mg and (ii) Kd of 1.1 mM, capacity of 84 nmol/mg. To assess
the molecular origin of the sarcolemmal calcium binding we treated
the membranes with a variety of enzymes. Protease or neuraminidase
treatment did not cause large changes in these parameters. Simultaneous
treatment with two different phospholipases C or the extraction of
the lipids with isopropanol resulted in a dramatic loss of the low-affinity
binding sites. These results, in association with previously defined
sarcolemmal phospholipid distribution, places the low-affinity binding
sites at the cytoplasmic leaflet. The physiological implication of
this localization as it pertains to cellular calcium exchange is
discussed.},
added-at = {2009-06-03T11:20:58.000+0200},
author = {Post, J. A. and Langer, G. A.},
biburl = {https://www.bibsonomy.org/bibtex/26a4500946e8ad7f676f9fbe60c496922/hake},
description = {The whole bibliography file I use.},
doi = {10.1007/BF00232054},
file = {Post_1992_49.pdf:Post_1992_49.pdf:PDF},
interhash = {edb3fbd3ad6dd5bd18c31bd1635ba96e},
intrahash = {6a4500946e8ad7f676f9fbe60c496922},
journal = {J. Membr. Biol.},
key = 237,
keywords = {1-Propanol, 1404340 Animals, Binding C, Calcium Calcium, Cell Cells, Compartmentation, Cultured, Dissection, Endopeptidases, Gov't, Lanthanum, Models, Myocardium, Neuraminidase, Newborn, Nifedipine, Nitrogen, Non-U.S. P.H.S., Phospholipase Phospholipids, Radioisotopes, Rats, Research Reticulu, Ryanodine, Sarcolemma, Sarcoplasmic Sites, Support, Terpenes, Thapsigargin, Theoretical, U.S. m,},
month = Jul,
number = 1,
pages = {49--57},
pmid = {1404340},
timestamp = {2009-06-03T11:21:26.000+0200},
title = {Sarcolemmal calcium binding sites in heart: I. Molecular origin in
"gas-dissected" sarcolemma.},
url = {http://dx.doi.org/10.1007/BF00232054},
volume = 129,
year = 1992
}