In this exercise, students purify cabbage phospholipase D in one step using a simple type of affinity chromatography and demonstrate its hydrolytic and transferase activities. Though cabbage phospholipase D is an old familiar face to lipid biochemists, it had proven extremely difficult to purify until a German group in 1992 reported using hydrophobic affinity chromatography to purify it in one easy step from a crude cabbage extract [see Biol. Chem. Hoppe-Seyler 373, 81-88 (1992)]. Briefly, the enzyme binds tightly to a hydrophobic matrix in the presence of calcium and elutes with EDTA. The enzyme hydrolyzes phospholipids between the headgroup alcohol and the phosphate group, in essence transferring the phosphatidyl group to water. What has proven more interesting is its ability to transfer phosphatidyl groups to other alcohols, producing novel phospholipids Nature never saw. For those not in the know, phospholipase D is also a hot item in the repertoire of mammalian lipid-dependent signaling mechanisms.

Preparation of Crude Cabbage Extract: Typically, I prepare the crude cabbage extract to save time, but there is no reason students couldn't do it. For reference, see Methods in Enzymology, Vol. XIV, pp. 208-211. The last time I did this I used a blender to blend 450 g of light green inner leaves of Savoy cabbage (small pieces) with 700 mL of water in three separate portions. Then I wrapped the mash in four layers of cheesecloth and squeezed to filter; the yield was about 700 mL of green juice. In several runs, I centrifuged the juice at 9,000 rpm for 10 minutes (Sorvall SS-34 rotor). I then heated the pooled supernatants in a microwave oven to 55 degrees. After three minutes, I cooled the mixture in an ice-water bath and centrifuged at 10,000 rpm for 25 minutes. To the supernatant, I added one-half volume of cold 95% ethanol, stored in the freezer overnight, and centrifuged the cloudy mixture the next day at 10,000 rpm for 10 minutes. I resuspended the pellet three times with 6 mL of water, centrifuging each time. The clear supernatant had a lot of activity. Students used it for the chromatography step.

Hydrophobic Affinity Chromatography: This procedure uses three buffers, all at pH 6.2: Buffer A is 30 mM PIPES, 50 mM calcium chloride; Buffer B is 10 mM PIPES, 30 mM calcium chloride; and Buffer C is 10 mM PIPES, 0.1 mM EDTA. Octyl-Sepharose is expensive but students only need 2 mL and it can be reused nearly indefinitely if washed with detergent after use (below). Students prepare a 2-mL minicolumn with a glass wool plug in, for example, a Pasteur pipet or a plastic dropping pipet with the top cut off and wash it with 5 mL of Buffer A. Then they pour the following over the column, collecting each run-through separately on ice: 2 mL of crude extract (above), 3 mL of Buffer A (4 times), 3 mL of Buffer B (2 times), and 3 mL of Buffer C (4 times), giving eleven fractions.

Students estimated protein in each fraction by mixing 0.3 mL with 3 mL of Bradford protein assay reagent and determining absorbance at 595 nm. Phospholipase D activity in the original extract and in each fraction was estimated by mixing one drop of sample with 0.3 mL of reaction mixture [0.5 mg/mL egg yolk phosphatidycholine, 0.3 mM sodium dodecyl sulfate, 0.1 mM methyl red, and 40 mM calcium chloride, adjusted (carefully, no buffer!) to pH 6.2] in a porcelain spot plate and incubating at room temperature for 10-15 minutes. Hydrolysis of phosphatidylcholine lowers the pH due to ionization of the new phosphate group, causing the indicator to change color from yellow to red. It's very simple but elegant. (Other indicators with color changes in this pH range might work as well, though I haven't been successful finding one.) Typically, the only fractions with activity are the original extract and the Buffer C eluates. Active fractions are pooled and saved in the refrigerator.

When the exercise is over, I collect all the octyl-Sepharose and wash it together with a detergent-NaOH-EDTA solution which strips most or all of the residual protein from it, then rinse it well with water and store in 33% ethanol in the refrigerator.

Phosphatidyl Transferase Activity: I prepared 50% solutions of ethanol, glycerol, and ethanolamine. This reaction is most easily done with conical glass tubes having teflon-lined screw caps. In four separate tubes, students mixed two drops of phospholipase D (either their purified samples or the original cabbage extract) with one drop of each 50% alcohol solution or water. They then added 15 drops of 2.5 mg/mL phosphatidylcholine in diethyl ether and mixed well for 30 minutes. After adding two drops of 1 M HCl and mixing, they centrifuged briefly to separate phases and analyzed 20 microliters of each ether layer by thin-layer chromatography on 5 x 10 cm Silica Gel 60 plates. The developing solvent was chloroform/methanol/acetic acid (65:25:10 by volume). Plates were first exposed to iodine vapors briefly to locate spots, then sprayed lightly with 0.2% ninhydrin in ethanol and heated in a 120-degree oven to locate primary amines.

For their write-up, students were asked to:

1. tabulate the protein assay results from their octyl-Sepharose columns and indicate the color of their assay mixture for each fraction,
2. draw the structures of the three alcohols used, of phosphatidylcholine, and of the products expected in each of their four reaction tubes,
3. draw a sketch of their TLC plate with spots identified as to whether they were stained by iodine or ninhydrin,
4. comment on the extent of reaction in each tube,
5. identify the ninhydrin-stained product (a naturally-occurring lipid),and
6. state which of the products has a new chiral center (one isomer of which is also a naturally-coourring lipid).

You could use any number of other alcohols as acceptor. The enzyme seems to prefer small primary alcohols, but experiment!

Revised 6/14/07