Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A sub 2 : A sup 13 C NMR study

Journal Article · Tue Aug 08 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00442a020· OSTI ID:5300504

Bhamidipati, S P; Hamilton, J A ^[1]

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1. 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), Journal Name: Biochemistry; (USA) Vol. 28:16; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALBUMINS
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON ISOTOPES
CHEMICAL REACTIONS
CHEMICAL SHIFT
DECOMPOSITION
ENZYMATIC HYDROLYSIS
ESTERS
EVEN-ODD NUCLEI
HEAVY WATER
HYDROGEN COMPOUNDS
HYDROLYSIS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPASE
LIPIDS
LYSIS
MAGNETIC RESONANCE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHOLIPIDS
PROTEINS
REACTION KINETICS
RESONANCE
SOLVOLYSIS
SPECTRA
STABLE ISOTOPES
WATER