A spectrophotometric assay for phospholipase D

doi:10.1016/0003-2670(94)00613-Q

Analytica Chimica Acta

Volume 304, Issue 2, 30 March 1995, Pages 249-254

https://doi.org/10.1016/0003-2670(94)00613-Q Get rights and content

Abstract

The preparation of phosphatidyl-p-nitrophenol and its use as a substrate in the non-enzyme based assay for the quantitative evaluation of phospholipase D activity is described. The solution of the substrate in Tris buffer pH 8 and Triton X-100 is mixed directly with an aliquot of a fermentation broth or a plant extract and the absorbance of the solution at 405 nm is read. The method is rapid, accurate and inexpensive. Application in the pH range 4–9 is possible.

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Cited by (58)

Immobilization of phospholipase D on macroporous SiO<inf>2</inf>/cationic polymer nano-composited support for the highly efficient synthesis of phosphatidylserine
2020, Enzyme and Microbial Technology
Novel nano-composites were prepared by coating epoxy resin-based cationic polymer in nano-thickness via in-situ curing on the nano-wall of macroporous SiO₂ with pore size of 0.5∼1 μm. By changing the thickness of polymer coating the specific surface area and porosity varied in range of 115∼74 m²/g and 90.4∼83.9 %, respectively. Through ion exchange phospholipase D (PLD, from Streptomyces sp) was efficiently immobilized on the nano-composites as support and the immobilized PLD was applied for the highly efficient synthesis of phosphatidylserine (PS). The loading amount of PLD on the nano-composited support reached to a maximum of 90.2 mg/g_support, 4 times as high as that on the pure macroporous silica. The specific activity of the immobilized PLD reached as high as 16,230 U/g_protein, while that of free PLD was 18,780 U/g_protein. Under a wide range of temperature and pH the stability and activity of the immobilized PLD were greatly improved as compared with the free ones. Under optimized conditions at 45 °C and pH 7.0, the PS yield reached as high as 96.2 % within 40 min. After 28 days storage the immobilized PLD retained 82.2 % of original activity, and after 12 cycling reuses it retained 79.3 % of PS yield, which indicated that the immobilized PLD exhibited good stability.
Efficient immobilization of phospholipase D on novel polymer supports with hierarchical pore structures
2019, International Journal of Biological Macromolecules
In this article, novel epoxy resin-based hierarchical porous polymers (HPSs) have been prepared through a non-sol-gel and template-free approach using crystalline trimethylolpropane (TMP) as porogen. The polymers exhibit dimensional stability and possess 3-dimentional interconnected multi-scale pores. In range of 50 μm~10 nm are ultra-macro-pore in between skeleton, macro-pore on skeleton and meso-pore in network, respectively. The porosity and specific surface area can be adjusted in range of 91.2–82.5% and 225–156 m²/g, respectively. Using three kinds of hierarchical porous polymers as supports phospholipase D (PLD) was effectively immobilized through physical adsorption. Owing to high porosity of the support and improvement of mass transfer the loading amount of PLD reached as high as 223 mg/g_support and the corresponding specific activity achieved up to 3.75 × 10³ U/g_support. Under optimized conditions and the phosphatidylserine (PS) yield reached 95.5% within 40 min at 45 °C. The immobilized PLD exhibited not only better storage stability and but also resistance to pH and thermal inactivation than free PLD. It was found that 73.5% of PS yield retained after 12 cycling reuses.
Deletion the C-terminal peptides of Vibrio harveyi phospholipase D significantly improved its enzymatic properties
2019, International Journal of Biological Macromolecules
A novel phospholipase D that originate from marine Vibrio harveyi (VhPLD) was recombinant expressed and biochemically characterized. Moreover, effects of C-terminal peptides on catalytic and interfacial binding properties of VhPLD were investigated by constructing two truncated mutants (VhPLD-Δ(472–483) and VhPLD-Δ(437–483)). Optimal reaction temperature and pH value for wild-type VhPLD (VhPLD-WT) was 45 °C and pH 8.0. However, optimal reaction temperature of VhPLD-Δ(437–483) increased to 50 °C. Meanwhile, catalytic efficiency (k_cat/K_M) of VhPLD-Δ(472–483) and VhPLD-Δ(437–483) to the 1,2-Dioctanoyl-sn-glycero-3-phosphatidyl-p-nitrophenol (PpNP) was 12.9 and 14.2 times higher than that of VhPLD-WT. However, when compare the catalytic efficiency between VhPLD-Δ(472–483) and VhPLD-Δ(437–483), no significant change can be found between the two mutants. These results strongly indicated that the C-terminal 12 amino acids (472–483) have important role on the activity of VhPLD. Effects of C-terminal peptides on the interfacial binding properties of VhPLD to different phospholipid monolayers were also investigated by using monolayer film technology. Results of the maximum insertion pressure (MIP) indicated that deletion the C-terminal segment of VhPLD improved its interfacial binding properties to different phospholipid monolayers.
Highly active and stable nanobiocatalyst based on in-situ cross-linking of phospholipase D for the synthesis of phosphatidylserine
2018, International Journal of Biological Macromolecules
Phospholipase D (PLD) was effectively immobilized on a ZnO nanowires/macroporous SiO₂ composite support through an in-situ cross-linking method. An anionic and long-chained bi-epoxy cross-linker was used by adsorbing on the surface of ZnO nanowires through static interaction before cross-linking. Under the fine control of in-situ cross-linking the immobilized PLD has loading amount as high as 113.7 mg/g_support, possessing high specific activity from 13,987 to 16,142 U/g_protein in all the range of loading amount. The immobilized PLD showed high activity and stability in catalyzing the conversion of phosphatidylcholine (PC) to phosphatidylserine (PS). The reaction conditions such as loading amount of PLD, substrate molar ratio, temperature, solution pH, and reaction time were optimized for the finding of best synthetic process. Under optimized conditions and the PS yield reached 94.8% within 40 min at 50 °C. The immobilized PLD exhibited not only better thermostability and resistance to pH inactivation than free PLD but also the greatly improved storage stability and reusability. It was found that 81.5% of initial activity retained after incubation at 4 °C for 60 days and that 80.4% of PS yield retained after 13 cycling reuses.
Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D
2017, Phytochemistry
Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca²⁺ binding sites. In vitro, the predominantly expressed α-type PLDs need 20–100 mM CaCl₂ for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP₂), plays a secondary role. In the present paper, we have studied the interplay between PIP₂ and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP₂-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca²⁺ activation. Mg²⁺ ions are able to replace Ca²⁺ ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca²⁺ ions in the absence, but not in the presence, of PIP₂. The presence of PIP₂ causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca²⁺ by Tb³⁺ ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation.
Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel β-sheets consisting of four β-strands each and loop regions that harbor two Ca²⁺ binding sites. Based on the findings of the MD simulation, one of the bound Ca²⁺ ions is coordinated by five amino acid residues. The second Ca²⁺ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP₂ reveal, in addition to the previously postulated PIP₂-binding site in the middle of the β-sheet structure, another PIP₂-binding site near the two Ca²⁺ ions, which is in accordance with the experimental interplay of PIP₂, Ca²⁺ and Mg²⁺ ions.
Purification, sequencing and characterization of Phospholipase D from Indian mustard seeds
2015, Phytochemistry
Citation Excerpt :
Octyl-Sepharose CL-4B and Source 15Q were products of GE Healthcare, Freiburg, Germany. PpNP was synthesized in our laboratory as described by D’Arrigo et al. (1995). 1,3-dioctanoyl-glycero-2-phosphocholine (1,3-8:0/8:0-PC), 1,3-dilauroyl-glycero-2-phosphocholine (1,3-12:0/12:0-PC), and 1,3-dimyristoyl-glycero-2-phosphocholine (1,3-14:0/14:0-PC) were produced according to Haftendorn et al. (2000).
Phospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant Indian mustard seeds was purified by Ca²⁺-mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete amino acid sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0 kDa, pI 5.39, maximum activity at pH 5.5–6.0 and Ca²⁺ ion concentrations ⩾60 mM), allowed the assignment of this enzyme to the class of α-type PLDs. The apparent kinetic parameters V_max and K_m, determined for the hydrolysis of phosphatidylcholine (PC) in an aqueous mixed-micellar system were 356 ± 15 μmol min⁻¹ mg⁻¹ and 1.84 ± 0.17 mM, respectively. Phosphate analogs such as NaAlF₄ and Na₃VO₄ displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2–10 mM CaCl₂. PLD was inactivated at temperatures >45 °C. The enzyme exhibited the highest activity toward PC followed by phosphatidylethanolamine and phosphatidylglycerol. PCs with short-chain fatty acids were better substrates than PCs with long fatty acid chains. Lyso-PC was not accepted as substrate.

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SpectrophotometryA spectrophotometric assay for phospholipase D

Abstract

Trends Anal. Chem.

Anal. Chim. Acta

Methods Enzymol.

Methods Enzymol.

J. Lipid Res.

Anal. Biochem.

Adv. Lipid Res.

Methods Enzymol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Methods Enzymol.

Biochim. Biophys. Acta

Spectrophotometry
A spectrophotometric assay for phospholipase D