Merck
  • Home
  • Search Results
  • Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

Phytochemistry (2015-01-27)
Miriam Payá-Milans, Mónica Venegas-Calerón, Joaquín J Salas, Rafael Garcés, Enrique Martínez-Force
ABSTRACT

The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

MATERIALS
Product Number
Brand
Product Description

Supelco
Hydrogen chloride – methanol solution, ~1.25 m HCl (T), for GC derivatization, LiChropur
Sigma-Aldrich
Hydrogen chloride solution, 1.0 M in diethyl ether
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Hydrochloric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., fuming, ≥37%, APHA: ≤10
Supelco
HEPES, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
HEPES, anhydrous, free-flowing, Redi-Dri, ≥99.5%
Sigma-Aldrich
Hydrochloric acid, puriss., 24.5-26.0%
Sigma-Aldrich
Hydrochloric acid, meets analytical specification of Ph. Eur., BP, NF, fuming, 36.5-38%
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Hydrochloric acid, 37 wt. % in H2O, 99.999% trace metals basis
Sigma-Aldrich
Hydrogen chloride solution, 2.0 M in diethyl ether
Sigma-Aldrich
Hydrogen chloride solution, 4.0 M in dioxane
Sigma-Aldrich
Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology
Supelco
Hydrochloric acid solution, volumetric, 0.1 M HCl (0.1N), endotoxin free
Sigma-Aldrich
Hydrochloric acid solution, 1.0 N, BioReagent, suitable for cell culture
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Supelco
Hydrogen chloride – ethanol solution, ~1.25 M HCl, for GC derivatization, LiChropur
Sigma-Aldrich
Hydrochloric acid solution, ~6 M in H2O, for amino acid analysis
Supelco
Hydrogen chloride – 2-propanol solution, ~1.25 M HCl (T), for GC derivatization, LiChropur
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Hydrochloric acid, semiconductor grade PURANAL (Honeywell 17823), fuming 37%, 37-38%
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Hydrochloric acid solution, SAJ first grade, 9.5-10.0%
Sigma-Aldrich
Hydrochloric acid solution, 0.02 M
Sigma-Aldrich
Hydrochloric acid solution, 12 M
Sigma-Aldrich
Hydrogen chloride – ethanol solution, 0.1 M in ethanol
Sigma-Aldrich
Hydrochloric acid solution, 1 M
Sigma-Aldrich
Hydrogen chloride – ethanol solution, 3% in ethanol