Abstract

The goal of this research is to understand the molecular mechanics of the interfacial activity of water-soluble lipolytic enzymes. A variety of physical techniques (primarily NMR spectroscopy, but also DSC, EM, SANS) combined with kinetic analyses will be used to investigate phospholipid interactions with phospholipase-A2 (Naja naja naja) and phospholipase-C (Bacillus cereus), and phospholipid/triglyceride interactions with porcine pancreatic lipase and colipase. Specific problems include the following: (i) How do phospholipid interfaces vary in different aggregate structures and are these variations relevant to phospholipase activity? (ii) What are the stoichiometry and properties of phospholipid binding to phospholipase-A2 and -C? (iii) Can a unified kinetic model be developed that quantitatively accounts for phospholipase rates toward substrate as monomers, micelles, detergent mixed micelles, and bilayer vesicles? (iv) Is surface/core partitioning of triglyceride in lecithin/triglyceride particles measurable, variable, and can it be correlated with lipase activity? Results should be generalizable to a wide range of lipid/protein systems.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026762-10
Application #
3274202
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-09-01
Project End
1989-11-30
Budget Start
1988-04-01
Budget End
1989-11-30
Support Year
10
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Boston College
Department
Type
Schools of Arts and Sciences
DUNS #
045896339
City
Chestnut Hill
State
MA
Country
United States
Zip Code
02467

  Publications

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

Showing the most recent 10 out of 43 publications