Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium.

J. M. Mullaney, S. H. Chueh, T. K. Ghosh, Donald Gill

Research output: Contribution to journalArticle

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Abstract

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.

Original languageEnglish (US)
Pages (from-to)13865-13872
Number of pages8
JournalThe Journal of biological chemistry
Volume262
Issue number28
StatePublished - Jan 1 1987

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Guanine Nucleotides
Guanosine Triphosphate
Calcium
Oxalates
Guanosine 5'-O-(3-Thiotriphosphate)
Adenosine Diphosphate
Guanylyl Imidodiphosphate
Membranes
Membrane Fusion
Vanadates
Guanosine
Diphosphates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{f653fdb3e4c94402b25f966dd272e82d,
title = "Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium.",
abstract = "The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3{\%} polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.",
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Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium. / Mullaney, J. M.; Chueh, S. H.; Ghosh, T. K.; Gill, Donald.

In: The Journal of biological chemistry, Vol. 262, No. 28, 01.01.1987, p. 13865-13872.

Research output: Contribution to journalArticle

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T1 - Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium.

AU - Mullaney, J. M.

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N2 - The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.

AB - The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.

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