Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney
To determine whether protein kinase C is present in the basolateral membrane of the renal proximal tubular cell, we performed experiments to ascertain whether specific binding of (/sup 3/H)phorbol 12,13-dibutyrate could be demonstrated in basolateral membranes isolated from canine kidney. Specific binding was demonstrable that was half maximal at between 10(-7) and 10(-8) M phorbol 12,13-dibutyrate. Binding was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting phorbol esters, but not by inactive phorbol esters, including 4 alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol 12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of submicromolar concentrations of free calcium, enhanced phosphorylation of several proteins demonstrable in autoradiograms of sodium dodecyl sulfate-polyacrylamide gels originating from membranes subsequently exposed to (gamma-32P)ATP for 30 s. Dephosphorylation of (/sup 32/P)phosphoproteins was observed in gels from membranes incubated with (gamma-32P)ATP over time. TPA-stimulated phosphorylation of one protein band with Mr 135,000 was quantitated and was found to increase as a function of (TPA). Half-maximal TPA-stimulated phosphorylation of this protein band occurred at slightly less than 10(-9) M TPA. Our findings are consistent with a role for protein kinase C-effected phosphorylation of basolateral membrane proteins in the mediation or modulation of hormonal actions in the proximal tubular cell.
- Research Organization:
- Washington Univ. School of Medicine, St. Louis, MO
- OSTI ID:
- 5230579
- Journal Information:
- Am. J. Physiol.; (United States), Journal Name: Am. J. Physiol.; (United States)
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
CELL MEMBRANES
PHOSPHORYLATION
PHORBOL ESTERS
BIOLOGICAL EFFECTS
PHOSPHOTRANSFERASES
ENZYME ACTIVITY
AUTORADIOGRAPHY
CHEMICAL BONDS
DOGS
IN VITRO
KIDNEYS
PHOSPHOLIPIDS
PHOSPHORUS 32
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARCINOGENS
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERS
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPIDS
MAMMALS
MEMBRANES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RADIOISOTOPES
TRANSFERASES
VERTEBRATES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)
550201 - Biochemistry- Tracer Techniques