Trafficking and function of a membrane enzyme, the vacuolar proton ATPase
The vacuolar H+-ATPase (V-ATPase) is an integral membrane complex that couples the hydrolysis of cytosolic ATP to the transmembrane pumping of protons. Its subunits are organised into the soluble domain V1 (subunits A-H) attached to an integral membrane domain V0 (subunits a, c and d). V1 contains three ATP hydrolysing catalytic sites, and the integral membrane domain is responsible for transport of H+. Each catalytic cycle of the ATPase hydrolyses 3 ATP molecules, but the number of protons translocated during each catalytic cycle is uncertain. The integral membrane subunit a is known to exist as isoforms a1, a2, a3 and a4, although the functional differences between these isoforms are uncertain. All the V-ATPase isoforms contain the same soluble domain subunits.
As part of an experimental programme aimed at developing new anti-cancer drugs directed against the V-ATPase, the expression and cellular distribution of V-ATPase isoforms in BxPC-3 pancreatic cancer cells is being investigated. Membranes were extracted from cultured cells and separated on a 0.2 M to 2.0 M continuous sucrose gradient by centrifugation overnight at 100,000 g in a swing-out rotor. Each gradient was eluted from the centrifuge tube as seven separate fractions. Protein content of each fraction was analysed by SDS-PAGE and immunoblotting with antibodies to the V-ATPase a1 and a3 subunit isoforms and to the V-ATPase soluble domain B subunit. Immunoblots for these proteins were compared to blots developed with antibodies to marker proteins for specific cellular membranes. Figure 1 (above) shows the corresponding regions of each immunoblot.
1(a) Comment on the distribution of V-ATPase isoforms between different cellular membranes.
1( Comment on the relative expression levels of the V-ATPase isoforms. Explain your reasoning.
The V-ATPase in the membrane sample isolated in sucrose gradient fraction 6 was solubilised in the detergent Triton X-100 and purified by size exclusion chromatography on a Superose-6 column. The total bed volume of the column was 30 ml and the void or excluded volume was 6 ml. The protein peak corresponding to the solubilised V-ATPase eluted after a volume of 8.6 ml of buffer had passed through the column. To calibrate the column, molecular mass standard proteins were separated on the column and their elution volumes recorded (Table 1). The purified ATPase was collected in a total of 2 ml, and a protein assay indicated a final concentration of 150 µg ml-1.
Table 1. Elution volumes for mass standard proteins during size exclusion chromatography on Superose-6
Molecular mass standard Molecular Mass (kDa) Elution volume (ml)
Blue Dextran 2000 4.5
Botulinum neurotoxin 900 7.4
Thyroglobulin 669 9.8
Apoferritin 443 13.0
β-Amylase 200 18.8
Albumin 66 26.9
2(a) Calculate the molecular mass of the detergent-solubilised V-ATPase complex.
2( Calculate the molar concentration of the purified V-ATPase.
Cyclic GMP (cGMP) has been proposed to inhibit V-ATPase activity. To investigate this, catalytic activity of the purified V-ATPase was measured spectrophotometrically as the time-dependent release of inorganic phosphate from the substrate ATP. Reaction volume was 1 ml, containing 15 µg V-ATPase. Rates were determined with or without 20 nM cGMP for different ATP concentrations (Table 2). Rates are expressed as µmol phosphate released per minute per mg protein.
Table 2. Rates of ATP hydrolysis for isolated membrane fractions.
[ATP] µM Control rate Rate with cGMP
20 0.28 0.09
30 0.39 0.13
50 0.57 0.19
100 0.87 0.29
200 1.20 0.40
400 1.43 0.48
600 1.58 0.53
3(a) Calculate the Km and Vmax values for the V-ATPase with and without cGMP.
3( Calculate the Kcat (maximum number of substrate molecules that can be hydrolysed per active site per minute) of the V-ATPase
3© Comment on the effect of cGMP.
Measurements using the fluorescent dye SNARF-1 indicate that the cytoplasmic pH in the BxPC-3 cells is 7.4. The free energy cost of moving one mole of protons (ΔGH+) against an unfavourable pH gradient of magnitude ΔpH is given by the following equation:
Gas constant, R = 8.31 Joules mol-1
4(a) Assuming that the free energy of ATP hydrolysis is 39.5 kJ mol-1 and that the ATP:H+ ratio of the V-ATPase is 3:10, calculate the maximum pH gradient that can be established by the V-ATPase across the lysosomal membrane, operating at 37oC. What is the pH in the lumen of the lysosome?
If isoforms of the V-ATPase show differential sensitivity to drug molecules it opens up the possibility of targeting therapies towards particular cell types, such as cancer cells. The V-ATPase activity (in µmol phosphate released per minute per mg protein) in sucrose density gradient fractions 2 and 6 was measured in the presence of the potent V-ATPase specific inhibitor bafilomycin A1 and in the presence of a novel drug candidate, Compound A. Activity rates in the presence of these inhibitors are given in Table 3.
Table 3: ATPase rate data in the presence of inhibitor compounds
Inhibitor concentration (nM) ATPase activity in membrane fraction 6 ATPase activity in membrane fraction 2
+ Bafilomycin + Compound A + Bafilomycin + Compound A
0 4.3 4.3 1.8 1.8
0.25 4.3 4.3 1.8 1.78
0.5 4.26 4.3 1.78 1.75
1 4.04 4.3 1.69 1.33
2 3.72 4.3 1.56 0.83
6 2.83 4.3 1.18 0.315
12 2.03 4.16 0.85 0.19
18 1.21 3.92 0.51 0.16
25 0.81 3.72 0.34 0.16
50 0.47 2.83 0.2 0.15
100 0.39 2.03 0.16 0.14
200 0.37 1.21 0.16 0.12
300 0.35 0.81 0.14 0.12
500 0.34 0.47
1000 0.34 0.39
2000 0.34 0.39
5(a) Determine the IC50 values for bafilomycin A1 and Compound A for both membrane samples. The IC50 represents the concentration of a drug that is required for 50% inhibition of an enzymatic reaction in vitro.
5( Comment on these values.
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