Synaptic Vesicle Protein 2 (SV2) does not hydrolyze ATP

Cell culture and transfection

HEK293T cell (ATCC, Manassas, VA) culture and transfection with Lipofectamine™ 2000 reagent (Invitrogen, Grand Island, NY) were performed following the manufacturer's protocol¹¹. To transfect one 15 cm plate of cells, 48 µg of plasmid DNA and 96 µl of Lipofectamine™ 2000 reagent were mixed with 3 ml of serum and antibiotic-free minimum essential media (MEM) (Invitrogen, Grand Island, NY). After incubation at room temperature for 5 min, the DNA and Lipofectamine™ 2000 reagent mixture was combined and allowed to sit for 20 min at room temperature. The transfection mixture was then added drop-wise to cells cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified incubator containing 5% CO₂. After 48 h, medium was removed, cells were harvested, and rinsed with ice-cold phosphate-buffered saline (PBS) two times. Cell pellets were stored at -80°C until use. Rat SV2A and SV2B were expressed with a C-terminal FLAG epitope (DYKDDDK) in the mammalian expression vector pIRES2-EGFP (Clontech, Mountain View, CA).

Purification of SV2-FLAG protein

Recombinant SV2A and SV2B -FLAG fusion proteins were purified using anti-FLAG M2 affinity agarose beads (Sigma; catalog number: A2220, mouse IgG1 monoclonal antibody) as described previously¹¹. Briefly, transfected cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Cell extracts were made by adding extraction buffer (150 mM potassium acetate (J.T. Baker, Phillipsburg, NJ), 10 mM HEPES-KOH (pH 7.4) (Sigma, St. Louis, MO), 1% Triton X-100 (Roche Applied Science, Indianapolis, IN), with 1× fresh protease inhibitor mixture added before each use (Roche Applied Science, Indianapolis, IN)) and incubated at 4°C for 1 h. After extraction, insoluble material was removed by centrifugation (Beckman, Indianapolis IN) at 19,000 × g for 20 min. The resulting extract was mixed with pre-equilibrated anti-FLAG M2 agarose beads (Sigma, St. Louis, MO) and incubated with agitation at 4°C for 3–4 h. The beads were washed four times with 20 volumes of extraction buffer. FLAG fusion protein was eluted from the beads with 3× FLAG peptide (Sigma, St. Louis, MO) in buffer containing 150 mM potassium acetate, 10 mM HEPES-KOH (pH 7.4) and 0.5% Triton X-100. Final preparations were checked by silver staining (Thermo, Waltham, MA) of SDS polyacrylamide gels (Bio-Rad, Hercules, CA) and immunoblot with anti-SV2 (mouse monoclonal antibody against an epitope located on the N-terminus of SV2)¹² or anti-FLAG antibodies (Sigma, catalog number: F3165, mouse IgG1 monoclonal antibody). Protein concentration was determined by comparison to bovine serum albumin (BSA) (Fisher, Pittsburgh, PA) standards in PAGE gels. This method was used because the 3× FLAG peptide in the eluate will affect solution-based protein assays. BSA standards, ranging from 0.5 to 4 µg were loaded on the same gel along with purified SV2-FLAG fusion proteins. Gels stained with coomassie blue were scanned with a Kodak Image Station 440CF (Kodak, Rochester, NY). Net intensities of protein bands were determined by Kodak Molecular Imaging software (Kodak, Rochester, NY) and protein concentration of recombinant SV2-FLAG was calculated according to BSA standards.

ATP hydrolysis reaction

ATP hydrolysis reactions were conducted at 25°C in a buffer consisting of 150 mM potassium acetate, 10 mM HEPES (pH 7.4), 2 mM ATP, 3 MgCl₂, 0.1% Triton X-100, 1 mM DTT and 10–20 µg/ml purified recombinant SV2-FLAG protein. The ATP hydrolysis reaction was set up on ice and recombinant SV2-FLAG protein was added last. Reaction mixtures were prepared on ice, and an aliquot of reaction was immediately withdrawn as the 0°C, 0 min sample. The reaction was shifted to 25°C, and at designated intervals (1 min, 2 min, 5 min and 10 min), aliquots were removed from the reaction for analysis by the malachite green assay described below. Duplicates were assayed for each time point and the average value of the duplicates was used for quantification.

Commercially available calf intestinal alkaline phosphatase (CIP) (New England Biolabs, Ipswich, MA) was used as a positive control for the ATP hydrolysis reaction. As a negative control, an aliquot of CIP was heated at 95°C for 20 min to inactivate the enzyme.

Malachite green assay for inorganic phosphate concentration

To prepare the malachite green solution, one volume of 4.2% ammonium molybdate (Sigma, St. Louis, MO) in 4 M HCl was added to 3 volumes of 0.045% malachite green (Sigma, St. Louis, MO). The solution was stirred at room temperature for a minimum of 30 min before being filtered through a 0.22 µm membrane (Millipore, Billerica, MA). Malachite green solutions were kept at 4°C in the dark. Before each assay, 0.01% Tween-20 was added to the malachite green solution.

50 µl aliquots of ATP hydrolysis reaction were added to 950 µl of malachite green solution and color was allowed to develop for 20 min at room temperature. Absorbance at 620 nm was measured in a cuvette with the malachite green solution as a blank in a SmartSpec3000 spectrometer (Bio-Rad, Hercules, CA). A 0.02 mM KH₂PO₄ solution (Fisher, Pittsburgh, PA) was used as an inorganic phosphate standard. Phosphate release in ATP hydrolysis reactions was quantified by comparison to inorganic phosphate standards.