Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane

Sara A. Westwood, J. Paul Luzio, David A. Flockhart, Kenneth Siddle

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a 'high Km' form, whereas the particulate activity had both 'high Km' and 'low Km' components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both 'high Km' and 'low Km' phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The 'low Km' enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes. The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.

Original languageEnglish (US)
Pages (from-to)454-466
Number of pages13
JournalBiochimica et Biophysica Acta - General Subjects
Volume583
Issue number4
DOIs
StatePublished - Apr 3 1979
Externally publishedYes

Fingerprint

Phosphoric Diester Hydrolases
Cell membranes
Cyclic AMP
Rats
Hepatocytes
Cell Membrane
Liver
Membranes
Immunosorbents
Recovery
Rabbits
Intracellular Membranes
Enzyme Activation
Subcellular Fractions
Centrifugation
Enzyme activity
Substrates
Enzymes
Immunoglobulins
Sheep

Keywords

  • Cyclic-AMP phosphodiesterase
  • Subcellular distribution: (Rat hepatocyte, Plasma membrane)

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Medicine(all)

Cite this

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane. / Westwood, Sara A.; Luzio, J. Paul; Flockhart, David A.; Siddle, Kenneth.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 583, No. 4, 03.04.1979, p. 454-466.

Research output: Contribution to journalArticle

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AB - The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a 'high Km' form, whereas the particulate activity had both 'high Km' and 'low Km' components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both 'high Km' and 'low Km' phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The 'low Km' enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes. The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.

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