Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

Keith R Johnson, S. Scott Panter, Ross G. Johnson

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.

Original languageEnglish (US)
Pages (from-to)367-376
Number of pages10
JournalBBA - Molecular Cell Research
Volume844
Issue number3
DOIs
StatePublished - Mar 21 1985
Externally publishedYes

Fingerprint

Cyclic AMP-Dependent Protein Kinases
Lenses
Phosphorylation
Membranes
Holoenzymes
Catalytic Domain
Molecular Weight
Gels
Affinity Labels
Cell Membrane
Intercellular Junctions
Sepharose
Gel Chromatography
Membrane Proteins
Peptide Hydrolases
Phosphotransferases
Adenosine Triphosphate
Peptides
Enzymes

Keywords

  • Lens membrane
  • Membrane phosphorylation
  • Protein kinase
  • cyclic AMP

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens. / Johnson, Keith R; Panter, S. Scott; Johnson, Ross G.

In: BBA - Molecular Cell Research, Vol. 844, No. 3, 21.03.1985, p. 367-376.

Research output: Contribution to journalArticle

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N2 - We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.

AB - We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.

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