Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles

I. Édes, E. G. Kranias

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Abstract

The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2+-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9±0.9 and 10.1±0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activitics were 18.1±1.0 and 8.0±0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.

Original languageEnglish
Pages (from-to)78-87
Number of pages10
JournalBasic Research in Cardiology
Volume85
Issue number1
DOIs
Publication statusPublished - Jan 1990

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Phosphatidylinositol 4,5-Diphosphate
Type C Phospholipases
Guinea Pigs
Cell Membrane
Membranes
Phosphatidylinositol Phosphates
Deoxycholic Acid
Enzymes
Phosphatidylinositols
Phosphoinositide Phospholipase C
Phosphatidylserines
Phosphatidylcholines
Detergents
Cations
Phospholipids
Membrane Proteins
Hydrolysis
Proteins

Keywords

  • guinea pig
  • heart
  • phospholipase C
  • polyphosphoinositides

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

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title = "Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles",
abstract = "The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2+-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07{\%} deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9±0.9 and 10.1±0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activitics were 18.1±1.0 and 8.0±0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.",
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N2 - The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2+-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9±0.9 and 10.1±0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activitics were 18.1±1.0 and 8.0±0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.

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