凝胶延长分析二型脂肪酸合成的方法

Srinivas Kodali Andrew Galgoci Sheo Singh Dr. Jun Wang Dr., jun_wang2@merck.com, Merck Research Laboratories Related Journal & Article InformationManuscript: 2006-01-00990C

Journal: Nature

Article Title: Platensimycin is a selective FabF inhibitor with potent antibiotic properties

Introduction

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability. The significant differences between bacteria and humans in organization, structure of enzymes, and the role played by fatty acids make this pathway an attractive target for antibacterial drug discovery (refs. 1,2). Two marketed antibacterial agents that target the FabI enzyme are triclosan (antiseptic) and isoniazid (an anti-Mycobacterium tuberculosis agent)(refs 3,4). Two natural products, cerulenin (ref. 5) and thiolactomycin (ref. 6) which selectively inhibit the condensation enzymes FabH and FabF/B, were discovered more than two decades ago. New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors (refs. 7,8).

Materials

Reagents

DTT was from Fisher (BP172-5); beta-mercaptoethanol was from Bio-Rad (161-0710). [2-14C]Malonyl-CoA (60 mCi/mmol, Perkin Elmer, NEC612), ACP (Sigma, A7303) was pretreated with 3 mM DTT on ice for 20 min, aliquoted and stored at -80 °C. Cerulenin and thiolactomycin were purchased from Sigma. Triclosan could be obtained from VWR, 003384. Urea, PVDF membrane, 10X Tris/Glycine buffer and native protein sample buffer were purchased from Bio-Rad.

Equipment

Gel electrophoresis apparatus, Phosphor Screen and PhosphorImager scanner

Time Taken

Two days. Day one to run FASII assay, resolve it on the gel and overnight transfer of protein onto PVDF membrane. Day two to expose the phosphorscreen to protein bound to the PVDF membrane and scan image using PhosphorImager scanner

Procedure

1) FASII gel-elongation assay was done by preincubating 0.25 - 2 µg of E. coli or S. aureus lysate with a serial dilution of inhibitors at room temperature for 20 min in 50 µl of buffer containing 100 mM sodium phosphate (pH 7.0), 5 mM EDTA, 1 mM NADPH, 1 mM NADH, 150 µM DTT, 5 mM ß-mercaptoethanol, 20 µM acetyl-CoA or n-octanoyl-CoA or any other acyl-CoA as needed, 4% DMSO, and 8 µM of ACP pretreated with DTT. 2) The reaction was initiated by addition of 10 µl of water-diluted [C-14]malonyl-CoA, which gave a final concentration of 4 µM malonyl-CoA. 3) The reaction was incubated at 37°C for 30 min for E. coli lysate and 60 min for S. aureus lysate. 4) After the reaction, 10 µl (plus 10 µl 2X native sample buffer) of each sample was directly applied to a l6% polyacrylamide gel containing a range of 0.4 M to 4 M urea as needed. 5) The gel was resolved and transfered to a polyvinylidene difluoride (PVDF) membrane, exposed to Phosphor Screen and visualized by using a PhosphorImager scanner.