The goal of this research is to understand the molecular mechanics of the interfacial activity of water-soluble lipolytic enzymes. We are using NMR, QLS, SANS, and fluorescence techniques in conjunction with kinetic analyses to investigate phospholipid interactions with the nonspecific phospholipase C (PLC) and the phosphatidylinositol-specific phospholipase C(PI-PLC) form Bacillus cereus. General key problems considered include the following. (i) How do phospholipid interfaces vary in different aggregate structures, and can these variations be correlated with phospholipase activity (i.e. can we explain """"""""interfacial activation"""""""", """"""""substrate inhibition"""""""", """"""""surface dilution"""""""", etc.)? (ii) What phospholipase C species? (iii) Can a unified kinetic model be developed that quantitatively accounts for phospholipase rates toward substrate as monomer, micelles, detergent mixed micelles, and bilayer vesicles? The results of these studies will allow us to understand the complex interfacial behavior of two bacterial PLC type enzymes. Since the bacterial nonspecific Plc has characteristics similar to at least on neutral mammalian PLC, the general phenomena and rationale we develop for the B. cereus enzymes may be extrapolated to the important mammalian enzymes. The latter play key roles in cellular activation including the production of inositol phosphates for intracellular Ca2+ mobilization and the generation of diglyceride for activation of protein kinase C.

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National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Boston College
Schools of Arts and Sciences
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Yang, Hongying; Roberts, Mary F (2004) Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D. Biochim Biophys Acta 1703:43-51
Zambonelli, Carlo; Casali, Monica; Roberts, Mary F (2003) Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities. J Biol Chem 278:52282-9
Yang, Hongying; Roberts, Mary F (2003) Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis. Protein Sci 12:2087-98
Oh, Mi-Kyung; Yang, Hongying; Roberts, Mary F (2003) Using O-(n-alkyl)-N-(N,N'-dimethylethyl)phosphoramidates to investigate the role of Ca2+ and interfacial binding in a bacterial phospholipase D. Biochim Biophys Acta 1649:146-53
Zambonelli, Carlo; Roberts, Mary F (2003) An iron-dependent bacterial phospholipase D reminiscent of purple acid phosphatases. J Biol Chem 278:13706-11
Yang, Hongying; Roberts, Mary F (2002) Cloning, overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D. Protein Sci 11:2958-68
Stieglitz, K A; Seaton, B A; Roberts, M F (2001) Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion. Biochemistry 40:13954-63
Geng, D; Baker, D P; Foley, S F et al. (1999) A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus. Biochim Biophys Acta 1430:234-44
Stieglitz, K; Seaton, B; Roberts, M F (1999) The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid. J Biol Chem 274:35367-74
Zhou, C; Horstman, D; Carpenter, G et al. (1999) Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface. J Biol Chem 274:2786-93

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