Trypsin-induced calcium efflux from sarcoplasmic reticulum

Evidence for the involvement of the (Ca2++Mg2+)-ATPase

Jun Ling Huang, Traci B. Topping, Zhaoping He, Brian Folsom, A. Dunker

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al., Membr. Biochem.5:49-75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5′-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.

Original languageEnglish (US)
Pages (from-to)193-205
Number of pages13
JournalJournal of Membrane Biology
Volume100
Issue number1
DOIs
StatePublished - Dec 1987
Externally publishedYes

Fingerprint

Ca(2+) Mg(2+)-ATPase
Sarcoplasmic Reticulum
Trypsin
Digestion
Adenosine Triphosphatases
Permeability
Calcium
Temperature
Carbodiimides
Membranes
Calcium-Transporting ATPases
Calcium Channels
Proteins

Keywords

  • calcium ATPase
  • sarcoplasmic reticulum
  • trypsin digestion

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biophysics

Cite this

Trypsin-induced calcium efflux from sarcoplasmic reticulum : Evidence for the involvement of the (Ca2++Mg2+)-ATPase. / Huang, Jun Ling; Topping, Traci B.; He, Zhaoping; Folsom, Brian; Dunker, A.

In: Journal of Membrane Biology, Vol. 100, No. 1, 12.1987, p. 193-205.

Research output: Contribution to journalArticle

Huang, Jun Ling ; Topping, Traci B. ; He, Zhaoping ; Folsom, Brian ; Dunker, A. / Trypsin-induced calcium efflux from sarcoplasmic reticulum : Evidence for the involvement of the (Ca2++Mg2+)-ATPase. In: Journal of Membrane Biology. 1987 ; Vol. 100, No. 1. pp. 193-205.
@article{b7bc73fe5014404e89d6947c3099fcea,
title = "Trypsin-induced calcium efflux from sarcoplasmic reticulum: Evidence for the involvement of the (Ca2++Mg2+)-ATPase",
abstract = "Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al., Membr. Biochem.5:49-75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5′-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.",
keywords = "calcium ATPase, sarcoplasmic reticulum, trypsin digestion",
author = "Huang, {Jun Ling} and Topping, {Traci B.} and Zhaoping He and Brian Folsom and A. Dunker",
year = "1987",
month = "12",
doi = "10.1007/BF02209150",
language = "English (US)",
volume = "100",
pages = "193--205",
journal = "Journal of Membrane Biology",
issn = "0022-2631",
publisher = "Springer New York",
number = "1",

}

TY - JOUR

T1 - Trypsin-induced calcium efflux from sarcoplasmic reticulum

T2 - Evidence for the involvement of the (Ca2++Mg2+)-ATPase

AU - Huang, Jun Ling

AU - Topping, Traci B.

AU - He, Zhaoping

AU - Folsom, Brian

AU - Dunker, A.

PY - 1987/12

Y1 - 1987/12

N2 - Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al., Membr. Biochem.5:49-75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5′-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.

AB - Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al., Membr. Biochem.5:49-75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5′-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.

KW - calcium ATPase

KW - sarcoplasmic reticulum

KW - trypsin digestion

UR - http://www.scopus.com/inward/record.url?scp=0023483157&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023483157&partnerID=8YFLogxK

U2 - 10.1007/BF02209150

DO - 10.1007/BF02209150

M3 - Article

VL - 100

SP - 193

EP - 205

JO - Journal of Membrane Biology

JF - Journal of Membrane Biology

SN - 0022-2631

IS - 1

ER -