Polyamine-phospholipid interaction probed by the accessibility of the phospholipid sn-2 ester bond to the action of phospholipase A2.
Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 1:1 and 12:1, respectively. Double-reciprocal plots of enzyme activity vs. PPHPG concentration revealed the enhancement to be due to increased apparent Vmax while the apparent Km was slightly increased. In the presence of 4 mM CaCl2 inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl2 polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4 mM CaCl2 reversed this inhibition and a twofold activation was observed at 10 microM spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.
D000818:Animals; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids; D011073:Polyamines / Q000494:pharmacology*; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
D010743:Phospholipids; D011073:Polyamines; D013096:Spermine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D013095:Spermidine