Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes
There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-(32P) lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-(32P)PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-(32P)phosphatidic acid (alkyl-(32P)PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-(32P)PA must be formed via PLD-catalyzed hydrolysis of alkyl-(32P)PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-(32P)phosphatidylethanol (alkyl-(32P)PEt). Formation of alkyl-(32P)PEt parallels that of alkyl-(32P)PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration.
- Research Organization:
- Schering-Plough Corporation, Bloomfield, NJ (USA)
- OSTI ID:
- 6758109
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 263:25
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LIPASE
ENZYME ACTIVITY
PHOSPHOLIPIDS
METABOLISM
BIOCHEMICAL REACTION KINETICS
CALCIUM
LEUKOCYTES
PHOSPHORUS 32
TRACER TECHNIQUES
ALKALINE EARTH METALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLESTERASES
DAYS LIVING RADIOISOTOPES
ELEMENTS
ENZYMES
ESTERASES
ESTERS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPIDS
MATERIALS
METALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
REACTION KINETICS
550501* - Metabolism- Tracer Techniques