Synthesis and translocation of Na+-K+-ATPase α-and β-subunits to plasma membrane in MDCK cells

A. K. Mircheff, J. W. Bowen, Samuel Chi-Hung Yiu, A. A. McDonough

Research output: Contribution to journalArticle

Abstract

Synthesis and translocation of Na+-K+-ATPase α-catalytic and β- glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into β-subunit was equal to that incorporated into α-subunit after 15 min of labeling. Because the ratio of total methionines in α- vs. β-subunit is ~5:1, these results suggest that β-subunit is synthesized in molar excess over α-subunit. Half of the newly synthesized β-subunit, likely unassembled units, were degraded by 60 min after labeling, while α-subunits were stable through 120 min after synthesis, suggesting α may be limiting for αβ-assembly. By 120 min the ratio of counts incorporated into α vs. β approached 5, which is predicted by a 1:1 ratio of α to β. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature β (β(i)) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature β (β(m)) became detectable after 30 min, and conversion of β(i) to β(m) was 90% complete at 120 min. A peak of labeled α-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled β(m)-subunit in this sample, suggesting movement as αβ-heterodimers.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume262
Issue number2 31-2
StatePublished - 1992
Externally publishedYes

Fingerprint

Madin Darby Canine Kidney Cells
Cell membranes
Adenosine Triphosphatases
Cell Membrane
Labeling
Intracellular Membranes
Sorbitol
Membranes
Immunoprecipitation
Methionine
Fractionation
Endoplasmic Reticulum
Catalytic Domain
Glycoproteins
Contamination
sodium-translocating ATPase
Population

Keywords

  • endoplasmic reticulum
  • Golgi complex
  • immunodetection
  • plasma membranes
  • sodium pump

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Synthesis and translocation of Na+-K+-ATPase α-and β-subunits to plasma membrane in MDCK cells. / Mircheff, A. K.; Bowen, J. W.; Yiu, Samuel Chi-Hung; McDonough, A. A.

In: American Journal of Physiology - Cell Physiology, Vol. 262, No. 2 31-2, 1992.

Research output: Contribution to journalArticle

@article{90320c72d42f4c2281988a52d5686f69,
title = "Synthesis and translocation of Na+-K+-ATPase α-and β-subunits to plasma membrane in MDCK cells",
abstract = "Synthesis and translocation of Na+-K+-ATPase α-catalytic and β- glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into β-subunit was equal to that incorporated into α-subunit after 15 min of labeling. Because the ratio of total methionines in α- vs. β-subunit is ~5:1, these results suggest that β-subunit is synthesized in molar excess over α-subunit. Half of the newly synthesized β-subunit, likely unassembled units, were degraded by 60 min after labeling, while α-subunits were stable through 120 min after synthesis, suggesting α may be limiting for αβ-assembly. By 120 min the ratio of counts incorporated into α vs. β approached 5, which is predicted by a 1:1 ratio of α to β. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature β (β(i)) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature β (β(m)) became detectable after 30 min, and conversion of β(i) to β(m) was 90{\%} complete at 120 min. A peak of labeled α-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled β(m)-subunit in this sample, suggesting movement as αβ-heterodimers.",
keywords = "endoplasmic reticulum, Golgi complex, immunodetection, plasma membranes, sodium pump",
author = "Mircheff, {A. K.} and Bowen, {J. W.} and Yiu, {Samuel Chi-Hung} and McDonough, {A. A.}",
year = "1992",
language = "English (US)",
volume = "262",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "2 31-2",

}

TY - JOUR

T1 - Synthesis and translocation of Na+-K+-ATPase α-and β-subunits to plasma membrane in MDCK cells

AU - Mircheff, A. K.

AU - Bowen, J. W.

AU - Yiu, Samuel Chi-Hung

AU - McDonough, A. A.

PY - 1992

Y1 - 1992

N2 - Synthesis and translocation of Na+-K+-ATPase α-catalytic and β- glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into β-subunit was equal to that incorporated into α-subunit after 15 min of labeling. Because the ratio of total methionines in α- vs. β-subunit is ~5:1, these results suggest that β-subunit is synthesized in molar excess over α-subunit. Half of the newly synthesized β-subunit, likely unassembled units, were degraded by 60 min after labeling, while α-subunits were stable through 120 min after synthesis, suggesting α may be limiting for αβ-assembly. By 120 min the ratio of counts incorporated into α vs. β approached 5, which is predicted by a 1:1 ratio of α to β. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature β (β(i)) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature β (β(m)) became detectable after 30 min, and conversion of β(i) to β(m) was 90% complete at 120 min. A peak of labeled α-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled β(m)-subunit in this sample, suggesting movement as αβ-heterodimers.

AB - Synthesis and translocation of Na+-K+-ATPase α-catalytic and β- glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into β-subunit was equal to that incorporated into α-subunit after 15 min of labeling. Because the ratio of total methionines in α- vs. β-subunit is ~5:1, these results suggest that β-subunit is synthesized in molar excess over α-subunit. Half of the newly synthesized β-subunit, likely unassembled units, were degraded by 60 min after labeling, while α-subunits were stable through 120 min after synthesis, suggesting α may be limiting for αβ-assembly. By 120 min the ratio of counts incorporated into α vs. β approached 5, which is predicted by a 1:1 ratio of α to β. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature β (β(i)) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature β (β(m)) became detectable after 30 min, and conversion of β(i) to β(m) was 90% complete at 120 min. A peak of labeled α-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled β(m)-subunit in this sample, suggesting movement as αβ-heterodimers.

KW - endoplasmic reticulum

KW - Golgi complex

KW - immunodetection

KW - plasma membranes

KW - sodium pump

UR - http://www.scopus.com/inward/record.url?scp=0026555312&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026555312&partnerID=8YFLogxK

M3 - Article

VL - 262

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 2 31-2

ER -