Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity.
Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis.
Koumanov
Kamen S
KS
Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Momchilova
Albena B
AB
Quinn
Peter J
PJ
Wolf
Claude
C
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D020404:Glycerophospholipids / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D013997:Time Factors; D014961:X-Ray Diffraction
D001095:Arachidonic Acids; D002518:Ceramides; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D020404:Glycerophospholipids; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D013109:Sphingomyelins; D002784:Cholesterol; D010741:Phospholipases A; D054467:Phospholipases A2