Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A sub 2 : A sup 13 C NMR study
- Boston Univ. School of Medicine, MA (USA)
A new approach to study phospholipase A{sub 2} mediated hydrolysis of phospholipid vesicles, using {sup 13}C NMR spectroscopy, is described. ({sup 13}C)Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A{sub 2} (Crotalus adamanteus). The {sup 13}C-labeled carboxyl/carbonyl peaks from the products (lyso-1-palmitoyl-phosphatidylcholine (LPPC) and palmitic acid (PA)) were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was {approximately} 50% ionized at pH {approximately} 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of {sup 13}C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.
- OSTI ID:
- 5300504
- Journal Information:
- Biochemistry; (USA), Vol. 28:16; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
Similar Records
Kinetic study of the action of snake venom phospholipase A/sub 2/ on human serum high density lipoprotein 3. [Crotalus adamanteus]
Interfacial catalysis by phospholipase A sub 2 : Evaluation of the interfacial rate constants by steady-state isotope effect studies
Related Subjects
LIPASE
NUCLEAR MAGNETIC RESONANCE
PHOSPHOLIPIDS
ENZYMATIC HYDROLYSIS
ALBUMINS
BIOCHEMICAL REACTION KINETICS
CARBON 13
CHEMICAL REACTIONS
CHEMICAL SHIFT
HEAVY WATER
NMR SPECTRA
CARBON ISOTOPES
DECOMPOSITION
ESTERS
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
HYDROLYSIS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPIDS
LYSIS
MAGNETIC RESONANCE
NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN COMPOUNDS
PROTEINS
REACTION KINETICS
RESONANCE
SOLVOLYSIS
SPECTRA
STABLE ISOTOPES
WATER
550201* - Biochemistry- Tracer Techniques