Evidence for an ATP-driven H+-pump in the plasma membrane of the bovine corneal epithelium

Viviana Torres-Zamorano, Vadivel Ganapathy, Mohamed M.H. Sharawy, Peter Reinach

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2·7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-β-d-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H+-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H+-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H+-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H+-pump activity was: ATP ≫ GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H+-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 μm NEM or NBD-Cl and 92% inhibited by 20 μm DCCD. The pH dependency of the NEM-sensitive ATPase activity showed a single optimum pH at 7·5. It is concluded that a major portion of the H+-pump activity in this membrane preparation is derived from the plasma membrane and that the properties of this plasma membrane H+-pump are similar to those of the vacuolar (V-type) H+-pumps described in various intracellular organelles and in the plasma membrane of other cell types.

Original languageEnglish (US)
Pages (from-to)269-277
Number of pages9
JournalExperimental eye research
Volume55
Issue number2
DOIs
StatePublished - Jan 1 1992

Fingerprint

Proton Pumps
Corneal Epithelium
Cholates
Adenosine Triphosphate
Cell Membrane
Membranes
Binding Sites
Inosine Triphosphate
Dicyclohexylcarbodiimide
Oligomycins
Cytidine Triphosphate
Hexosaminidases
Acridine Orange
Uridine Triphosphate
Vanadates
Azides
Guanosine Triphosphate
Organelles
Adenosine Triphosphatases
Chromatography

Keywords

  • V-type H-ATPase
  • corneal epithelium
  • intracellular pH
  • proton pump

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Evidence for an ATP-driven H+-pump in the plasma membrane of the bovine corneal epithelium. / Torres-Zamorano, Viviana; Ganapathy, Vadivel; Sharawy, Mohamed M.H.; Reinach, Peter.

In: Experimental eye research, Vol. 55, No. 2, 01.01.1992, p. 269-277.

Research output: Contribution to journalArticle

Torres-Zamorano, Viviana ; Ganapathy, Vadivel ; Sharawy, Mohamed M.H. ; Reinach, Peter. / Evidence for an ATP-driven H+-pump in the plasma membrane of the bovine corneal epithelium. In: Experimental eye research. 1992 ; Vol. 55, No. 2. pp. 269-277.
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N2 - In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2·7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-β-d-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H+-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H+-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H+-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H+-pump activity was: ATP ≫ GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H+-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 μm NEM or NBD-Cl and 92% inhibited by 20 μm DCCD. The pH dependency of the NEM-sensitive ATPase activity showed a single optimum pH at 7·5. It is concluded that a major portion of the H+-pump activity in this membrane preparation is derived from the plasma membrane and that the properties of this plasma membrane H+-pump are similar to those of the vacuolar (V-type) H+-pumps described in various intracellular organelles and in the plasma membrane of other cell types.

AB - In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2·7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-β-d-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H+-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H+-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H+-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H+-pump activity was: ATP ≫ GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg2+ with Mn2+ did not change the acidification rate but Co2+ only partly activated whereas Ca2+ and Zn2+ were ineffective as activators. The H+-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 μm NEM or NBD-Cl and 92% inhibited by 20 μm DCCD. The pH dependency of the NEM-sensitive ATPase activity showed a single optimum pH at 7·5. It is concluded that a major portion of the H+-pump activity in this membrane preparation is derived from the plasma membrane and that the properties of this plasma membrane H+-pump are similar to those of the vacuolar (V-type) H+-pumps described in various intracellular organelles and in the plasma membrane of other cell types.

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