Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens.
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.
Johnson
K R
KR
Panter
S S
SS
Johnson
R G
RG
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D007908:Lens, Crystalline / Q000201:enzymology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases