Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin
- Cornell Univ. Medical College, New York, NY (USA)
Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells. The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from spingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with (3H)serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate.
- OSTI ID:
- 6511607
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 265:25; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
PHOSPHOLIPIDS
BIOSYNTHESIS
SPHINGOMYELINS
METABOLISM
AUTORADIOGRAPHY
DOSE-RESPONSE RELATIONSHIPS
LEUKEMIA
MAN
PHOSPHATES
PHOSPHORUS 32
PHOSPHOTRANSFERASES
SERINE
TIME DEPENDENCE
TRITIUM COMPOUNDS
TUMOR CELLS
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DISEASES
ENZYMES
ESTERS
HEMIC DISEASES
HYDROGEN COMPOUNDS
HYDROXY ACIDS
IMMUNE SYSTEM DISEASES
ISOTOPES
LIGHT NUCLEI
LIPIDS
MAMMALS
NEOPLASMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PRIMATES
RADIOISOTOPES
SYNTHESIS
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques
550901 - Pathology- Tracer Techniques