Materials and Reagents

Ultracentrifuge tube (Beckman Coulter, catalog number: 349623 )
Cuvette (100 µl) (Beckman Coulter, catalog number: 523270 )
Note: This product has been discontinued.
Arabidopsis thaliana ecotype Col-0
Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14128-04 )
Phenylmethylsulfonyl fluoride (PMSF) (NACALAI TESQUE, catalog number: 273-27 )
Leupeptin (Wako Pure Chemical Industries, catalog number: 126-03754 )
MOPS (NACALAI TESQUE, catalog number: 23415-54 )
Oligomycin (Sigma-Aldrich, catalog number: 75351 )
Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
Ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA) (Dojindo Molecular Technologies, catalog number: N001-10 )
Sodium fluoride (NaF) (NACALAI TESQUE, catalog number: 31420-82 )
Tris (NACALAI TESQUE, catalog number: 35406-91 )
2-(N-morpholino)ethanesulfonic acid (MES) (NACALAI TESQUE, catalog number: 21623-26 )
Magnesium sulfate (MgSO₄) (Wako Pure Chemical Industries, catalog number: 131-00405 )
Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
Potassium nitrate (KNO₃) (Wako Pure Chemical Industries, catalog number: 160-04035 )
Ammonium molybdate (Wako Pure Chemical Industries, catalog number: 016-06902 )
Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
ATP (NACALAI TESQUE, catalog number: 10406-61 )
Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
SDS (NACALAI TESQUE, catalog number: 31607-65 )
Sodium molybdate (Wako Pure Chemical Industries, catalog number: 196-02472 )
Sulfuric acid (H₂SO₄) (Wako Pure Chemical Industries, catalog number: 192-04696 )
1-amino-2-naphthol-4-sulfonic acid (ANSA) (NACALAI TESQUE, catalog number: 02212-12 )
Sodium bisulfite (NaHSO₃) (Wako Pure Chemical Industries, catalog number: 190-01375 )
Sodium sulfate (Na₂SO₄) (Wako Pure Chemical Industries, catalog number: 192-03415 )
Potassium dihydrogen phosphate (KH₂PO₄) (Wako Pure Chemical Industries, catalog number: 169-04245 )
DTT stock solution (see Recipes)
Protease inhibitor solution (see Recipes)
Oligomycin solution (see Recipes)
Homogenization buffer (see Recipes)
2x ATPase buffer (see Recipes)
ATPase reaction buffer (see Recipes)
ATP solution (see Recipes)
Vanadate solution (see Recipes)
Stop solution (see Recipes)
ANSA solution (see Recipes)
Pi standard stock solution (see Recipes)

Equipment

Mortar (90 mm diameter) and pestle
Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-307 )
Ultracentrifuge (Beckman Coulter, model: OptimaTM TLX )
Vortex (Scientific Industry, model: SI-0286 )
Heat block (TAITEC, model: e-ThermoBucket ETB )
Spectrophotometer (Beckman Coulter, model: DU 730 )
Note: This product has been discontinued.

Procedure

Preparation of microsomal membranes
1. Grow Arabidopsis thaliana in soil for 3 weeks at 23 °C under white light (50 µmol photons m^-2 s^-1) with a 16-h-light/8-h-dark cycle.
2. Homogenize rosette leaves (about 100 mg) with a mortar and pestle in 2 ml ice-cold homogenization buffer and keep on ice.
3. Centrifuge the homogenate at 13,000 x g for 10 min at 4 °C.
4. Ultracentrifuge the supernatant at 100,000 x g for 1 h at 4 °C.
5. Resuspend the pellet in 100 μl ice-cold homogenization buffer by pipetting up and down.
6. Quantify the protein concentration by Bradford assay (Bradford, 1976).
7. Protein concentration is adjusted to 0.45 µg/µl with homogenization buffer.
  Note: Keep microsomal membranes on ice until use.
Measurement of vanadate-sensitive ATP hydrolytic activity
1. Mix 100 μl of microsomal membranes with 100 μl ATPase reaction buffer, split the mixture in two tubes, 90 µl each (20 µg protein), and keep on ice.
2. To determine vanadate-sensitive ATPase activity, add 2 μl vanadate solution to one tube and an equal volume of 1x ATPase buffer to the other tube.
3. Add 10 μl ATP solution and gently vortex.
4. Incubate at 30 °C for 30 min. Gently vortex once after 15 min of incubation.
5. Add 1 ml stop solution.
6. Add 50 μl ANSA solution and gently vortex.
7. Incubate at 24 °C for 30 min. Gently vortex once after 15 min of incubation.
8. Measure absorption at 750 nm by a spectrophotometer using a cuvette.
  Note: Do not centrifuge the samples.
Preparation of Pi standard curve
1. Prepare Pi dilution series as shown in Table 1.
  
  Table 1. Template for the preparation of the Pi standard curve
2. Add 50 μl ATPase reaction buffer and 1 ml stop solution.
3. Add 50 μl ANSA solution and incubate at 24 °C for 30 min. Gently vortex once after 15 min of incubation.
4. Measure absorption at 750 nm using a cuvette and make a standard curve (Figure 1).

Data analysis

A typical Pi standard curve is shown in Figure 1. Calculate Pi content of samples using the standard curve. Vanadate-sensitive ATP hydrolytic activity is determined by subtracting Pi content in the presence of vanadate from that in the absence of vanadate, and is expressed as nmol Pi/h/mg of protein.

Calculate a slope of Pi standard curve.
A₇₅₀ = 0.0038 (Pi content [nmol]) + 0.0088
Determine Pi content of samples from the slope.
(Pi content [nmol]) = (A₇₅₀ - 0.0088)/0.0038
Subtract Pi content in the presence of vanadate from that in the absence of vanadate.
Divide by the amount of protein (mg), and reaction time (h). The following is an example of calculation.
Pi content in the absence of vanadate = 10 nmol
Pi content in the presence of vanadate = 5 nmol
Reaction time = 0.5 h (30 min)
Amount of protein = 0.02 mg (20 µg)
Vanadate-sensitive ATP hydrolytic activity (nmol Pi/h/mg of protein)
= ([Pi content in the absence of vanadate] - [Pi content in the presence of vanadate])/(reaction time)/(Amount of protein)
= (10 - 5)/0.5/0.02 = 500 nmol Pi/h/mg of protein

Figure 1. Standard curve generated by using known amounts of Pi. The absorption at 750 nm was measured.

Recipes

DTT stock solution
1 M DTT in sterile water
Store at -20 °C in small aliquots
Protease inhibitor solution
200 mM PMSF and 4 mM leupeptin in DMSO
Store at 4 °C in small aliquots
Oligomycin solution
5 mg/ml oligomycin in DMSO
Store at -20 °C in small aliquots
Homogenization buffer
50 mM MOPS-KOH (pH 7.5)
100 mM NaCl
2.5 mM EDTA
10 mM NaF
5 mM DTT
1 mM PMSF
20 μM leupeptin
Store at 4 °C. Add NaF, DTT, PMSF, and leupeptin just before use
2x ATPase buffer
60 mM Tris-MES (pH 6.5)
6 mM MgSO₄
100 mM KCl
200 mM KNO₃
Store at 4 °C
ATPase reaction buffer (freshly prepared)
2x ATPase buffer supplemented with:
1 mM ammonium molybdate
10 μg/ml oligomycin
0.1% (w/w) Triton X-100
0.5 mM PMSF
10 μM leupeptin
ATP solution
20 mM ATP in 1x ATPase buffer
Solution should be aliquoted in small volumes to avoid freeze-thawing and can be stored at -80 °C for at least 6 months
Vanadate solution (freshly prepared)
10 mM sodium orthovanadate in 1x ATPase buffer
Stop solution (freshly prepared)
1.3% (w/v) SDS
0.25% (w/v) sodium molybdate
0.3 N H₂SO₄
ANSA solution (freshly prepared)
0.125% (w/v) 1-amino-2-naphthol-4-sulfonic acid (ANSA)
15% (w/v) NaHSO₃
1% (w/v) Na₂SO₄
Pi standard stock solution (freshly prepared)
5 mM KH₂PO₄

Acknowledgments

This protocol is adapted from Okumura et al. (2016). This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (15H05956 and 15H04386 to T.K.) and by a Grant-in-Aid for JSPS fellows (253307 to M.O.).

References

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2): 248-254.
Kinoshita, T., Nishimura, M. and Shimazaki, K. (1995). Cytosolic concentration of Ca²⁺ regulates the plasma membrane H⁺-ATPase in guard cells of fava bean. Plant Cell 7(8): 1333-1342.
Okumura, M., Inoue, S., Kuwata, K. and Kinoshita, T. (2016). Photosynthesis activates plasma membrane H⁺-ATPase via sugar accumulation. Plant Physiol 171(1): 580-589.
Shimazaki, K. I. and Kondo, N. (1987). Plasma membrane H⁺-ATPase in guard-cell protoplasts from Vicia faba L. Plant Cell Physiol 28(5): 893-900.
Uemura, M. and Yoshida, S. (1986). Studies on freezing injury in plant cells: II. Protein and lipid changes in the plasma membranes of Jerusalem artichoke tubers during a lethal freezing in vivo. Plant Physiol 80(1): 187-195.