title: Substrate efflux propensity is the key determinant of Ca2+-independent phospholipase A-β (iPLAβ)-mediated glycerophospholipid hydrolysis.
abstract: The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca(2+)-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.
authors.affiliation: From the Departments of Biochemistry and Developmental Biology and.
authors.forename: Krishna Chaithanya
authors.initials: KC
authors.lastname: Batchu
authors.affiliation: From the Departments of Biochemistry and Developmental Biology and.
authors.forename: Kati
authors.initials: K
authors.lastname: Hokynar
authors.affiliation: Biomedicine, Faculty of Medicine, University of Helsinki, Helsinki 00014, Finland.
authors.forename: Michael
authors.initials: M
authors.lastname: Jeltsch
authors.affiliation: Biomedicine, Faculty of Medicine, University of Helsinki, Helsinki 00014, Finland.
authors.forename: Kenny
authors.initials: K
authors.lastname: Mattonet
authors.affiliation: From the Departments of Biochemistry and Developmental Biology and pentti.somerharju@helsinki.fi.
authors.forename: Pentti
authors.initials: P
authors.lastname: Somerharju
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D057075:Enzyme Assays; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D020404:Glycerophospholipids / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006706:Homeostasis / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008823:Micelles; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology*; D054512:Phospholipases A2, Calcium-Independent / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D013379:Substrate Specificity
chemical_list: D020404:Glycerophospholipids; D008051:Lipid Bilayers; D008823:Micelles; D011994:Recombinant Proteins; D054512:Phospholipases A2, Calcium-Independent
keywords: Homeostasis; Mass Spectrometry (MS); Membrane; Membrane Bilayer; Phospholipase A