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J Biol Chem, Vol. 274, Issue 50, 35367-35374, December 10, 1999

The Role of Interfacial Binding in the Activation of Streptomyces chromofuscus Phospholipase D by Phosphatidic Acid^*

Kim Stieglitz, Barbara Seaton, and Mary F. Roberts^§¶

From the  Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118 and the ^§ Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH  7 in the presence of EGTA that is weakened as Ca²⁺ or Ba²⁺ are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba²⁺ was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA·Ca²⁺ surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca²⁺ modulation of PA·PLD binding involves protein aggregation that may be the critical interaction for activation.

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^* This work was supported by National Institutes of Health Grant GM 26762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Fax: 617-552-2705; E-mail: mary.roberts@bc.edu.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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