Summary
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 activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV max while the apparentK m was slightly increased. In the presence of 4mm 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 4mm CaCl2 reversed this inhibition and a twofold activation was observed at 10 μm 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.
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Thuren, T., Virtanen, J.A. & Kinnunen, P.K.J. Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2. J. Membrain Biol. 92, 1–7 (1986). https://doi.org/10.1007/BF01869010
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DOI: https://doi.org/10.1007/BF01869010