In situ phosphorylation of proteins in MCTs microdissected from rat kidney: Effect of AVP

Journal Article · Fri Apr 01 00:00:00 EST 1988 · American Journal of Physiology; (USA)

OSTI ID:6724807

Homma, S; Gapstur, S M; Yusufi, N K; Dousa, T P ^[1]

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1. Mayo Clinic and Foundation, Rochester, MN (USA)

Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, the authors developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, semipermeabilization, increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with {gamma}-({sup 32}P)ATP resulted in corporation of {sup 32}P{sub i} into two major protein bands detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in {sup 32}P{sub i} incorporation into multiple protein bands. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

• OSTI ID: 6724807
• Journal Information: American Journal of Physiology; (USA), Vol. 254:4; ISSN 0002-9513
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PROTEINS
PHOSPHORYLATION
SAPONINS
DOSE-RESPONSE RELATIONSHIPS
TUBULES
PERMEABILITY
VASOPRESSIN
BIOLOGICAL EFFECTS
AMP
ATP
CELL MEMBRANES
ELECTROPHORESIS
KIDNEYS
PHOSPHORUS 32
SUBSTRATES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
GLYCOSIDES
HORMONES
ISOTOPES
LIGHT NUCLEI
MEMBRANES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HORMONES
PHOSPHORUS ISOTOPES
PITUITARY HORMONES
RADIOISOTOPES
551001* - Physiological Systems- Tracer Techniques