A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2

Abstract

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Secretory Phospholipases A2 in Plants

“…In animals, a correct and functional sPLA 2 from Bothrops diporus was produced without any extra extension at the N-terminus (Yunes Quartino et al, 2012). Using the ubiquitin/deubiquitinase system, in the latter case, it was clearly shown that the recombinant protein had the same interfacial catalytic profile when compared to the native one (Yunes Quartino et al, 2015).…”

“…It is known that, in contrast to the seven disulfide bonds present in porcine pancreas enzyme ( Pp sPLA 2 ), all five disulfide bonds of Bv sPLA 2 are essential for conformational stability and contribute to the activity (Welker et al, 2011). In the case of bacterial sPLA 2 from Streptomyces violaceoruber , it possesses only two disulfide bridges (Sugiyama et al, 2002) which were sufficient to be active comparable to animal or plant sPLA 2 s (Yunes Quartino et al, 2015).…”

“…Several kinetics studies on pancreatic as well as snake venoms and plants phospholipases have been reported in which lipid phase transition, lipid membrane curvature, and composition may modulate the lipolysis (Wilschut et al, 1978; Bell and Biltonen, 1989; Bell et al, 1996; Leidy et al, 2004). However, it should not be forgotten, that sPLA 2 has optimum of lipid packing for hydrolysis, i.e., that some enzymes have the ability to hydrolyze lipid in a low packing organization (low lateral pressure in lipid monolayers more compatible with micelles) but others also have optimum condition of hydrolysis at high lateral pressure in monolayers compatible with liposomes or biological membranes (Ramirez and Jain, 1991; Yunes Quartino et al, 2015).…”

“…; from microorganisms as bacteria and yeasts, as components of pancreatic juices, where it occurs abundantly and has a digestive role; arthritic synovial fluid; and in many different mammalian tissues (Valentin et al, 1999; Schaloske and Dennis, 2006; Burke and Dennis, 2009; Murakami et al, 2010, 2011). Additionally, for the first time, we have recently described the interfacial properties of purified recombinant sPLA 2 s from Streptomyces violaceoruber (Yunes Quartino et al, 2015) and from Glycine max (Mariani et al, 2012, 2015b), i.e., the optimal surface lipid packing conditions (interfacial quality) in which a sPLA 2 can hydrolyze phospholipid in an organized membrane. This point, no less important for interfacial enzymes, was also addressed comparatively in the present review.…”

Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

“…Despite its medical relevance, the venom of B. diporus is still poorly characterized. Only a few proteins have been cloned, isolated and/or biochemically or functionally characterized, including the PLA 2 molecules, Myo-II (AFJ79209), s PLA 2 -I (AFJ79207), sPLA 2 -II (AFJ79208), svPLA 2 (C0HJP9) [ 11 , 12 , 13 , 14 , 15 , 16 ], and the C -type lectin-like protein SL1_BOTDP (C0HJQ0). However, a detailed view of the venom proteome is still missing.…”