Bacterial Energetics and Membranes

Bacterial Energetics and Membranes

Abstract
The Mg2+/Ca2+ ATP synthase present in all bacterial membranes, particularly E. coli, couples ATP synthesis to the proton (H+) gradient produced by the ETC, a process known as oxidative phosphorylation. The gradient acts to power the ATPase, so that it may phosphorylate ADP to produce ATP. The reverse reaction of this process, or hydrolysis of ATP into ADP and Pi, may be used to observe ATPase activity when the resulting Pi is quantitatively measured. In addition, the ETC activity may be observed when there is a measurable level of oxygen depletion as substrates are oxidized for their electrons that O2 accepts while being reduced to water. Here, we focus on Mg2+/Ca2+ ATPase magnesium dependence, its sensitivity to inhibition by DCCD, as well as ETC activity in the presence of NADH and malonate, and inhibition by KCN.
Introduction
Producing ATP is an important feature of all living cells. There are various ATP synthetases that utilize different methods to achieve ATP production. However, the need of an ion gradient to power ADP phosphorylation is central to all ATPases. Here, we focus on bacterial cell membranes, particularly E. coli, as they possess a unique Mg2+/Ca2+ ATPase. Several ATPase assays containing Pi, Mg2+ or no Mg2+, and DCCD were conducted where the reverse reaction -ATP hydrolysis into ADP and Pi- is used as an approximate measure of ATPase activity and dependence upon Mg2+, as well as the effects of inhibition by DCCD on Pi production. The ion gradient needed to power this ATPase will also be of importance as we determine what substrates the ETC preferentially oxidizes to produce the necessary H+ ion gradient. An oxygen consumption assay was employed with the use of an oxygen electrode attached to a pH meter. The electrode contained polarized silver electrodes and an electrolyte of chloride ions, separated from the treatments by a gas-permeable membrane. As oxygen diffuses through the membrane and is reduced to form OH- ions at the silver cathode, the anode oxidizes chloride ions to produce AgCl and an electron that is used for the reduction process at the cathode. The amount of Cl- oxidized and O2 reduced, is directly proportional to the amount of oxygen present outside the membrane. In each of the five treatments - NADH, NADH + Malonate, and NADH + KCN- the levels of oxygen will be recorded over a 20 minute span and will be used to indicate approximate ETC activity during ‘growth’ on various substrates and inhibition by KCN

Materials and Methods
Five separate assays were conducted, four pertaining to ATPase activity and Pi generation, and one specific to ETC activity. Assay A tubes 1-5 contained the following: 0.78 ml of 50 mM Tris-sulfate buffer, pH 7.8 containing 1 mM EDTA; 75 µl of 0.04 M MgCl2; and 100 µl of 10mg/ml E. coli membranes. Tubes A1-4 were mixed with a vortex and incubated at 37º C for 2 min while 50 µl 0.1 M ATP was added. At 2.5 minutes intervals for ten minutes, each tube was sequentially given 1 ml of 10% TCA, and subsequently put on ice. Assay B contained the same treatments, with the exception of MgCl2 omitted and substituted with 75 µl of H2O.
Assay C tubes 1-6 were given the following: 0.79 ml of 50 mM Tris-sulfate buffer, pH 7.8; 50 µl 0.04 M MgCl2; and 100 µl of 10mg/ml E. coli membranes. 10 mM DCCD was diluted with methanol to achieve the following concentrations and added to C1-6; 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, and 0 mM (10 µl of methanol) in C6. Tubes C1-6 were mixed and incubated at 37º C for 5 min before 50 µl ATP was added. The treatments were incubated for another 10 min upon which, the reactions were stopped with 1 ml TCA and put on ice.
Tubes from assay A, B, and C were centrifuged and 0.250 ml from the resulting supernatant was placed in 16 new tubes with 0.5 ml acid-molybdate and 1.725 ml H2O before 25 µl ANSA reagent was added. After 10 minutes, the absorbance readings were taken at 660 nm.
The fourth assay was conducted using 2.0 ml total volume of: 0.5 ml acid-molybdate; 25 µl ANSA reagent; and increasing amounts of Pi with decreasing volume of H2O to reach 2.0 ml final volume.
A final assay pertaining to the oxygen consumption, or ETC activity, was demonstrated with respiratory particles extracted from E. coli membranes. The oxygen electrode -and pH meter- apparatus was demonstrated with several treatments containing the following: buffer, E.coli membranes, NADH, NADH + Malonate, and NADH + KCN. Each sample was given varying amounts of H2O in-conjunction with the specified treatment (refer to Table 1.). The O­2 content in each sample was recorded over a 20 minute span at 760 mmHg.
Results
Previous experimentation has shown that bacterial membranes contain an Mg2+/Ca2+ ATP synthase (MCIM 391 Lab Manual, 2013) that produces ATP and pumps protons outside of the cytoplasm. An assay was completed in which increasing aliquots (0.1 ml) of inorganic phosphate (Pi) was mixed with ANSA and the absorbance’s read at 660nm. Upon mixing the ANSA reagent into each tube, a blue colour was observed that gradually became darker with the presence of more Pi. The absorbance values increased from 0.000 to 0.592 in tube 1 to tube 6, respectively, in a linear fashion. A standard curve was created from this data (Fig. 1.) in which approximate Pi production, or ATPase activity, was calculated for ATPase assays A and B.
Two ATPase assays were completed in which one contained 0.075 ml 0.04 M Mg2+ (tubes A1-5) while the other treatment omitted the Mg2+ and substituted it with 0.075 ml of H2O (tubes B1-5). The absorbance values for each tube, A1-4 and B1-4, were read at 660nm at 2.5 minute intervals for 10 minutes. While both assays showed increasing absorbance values during the 10 minute span, ranging from 0.027-0.100 (tube A1-4) and 0.025-0.042 (tube B1-4), tubes A1-4 showed a larger increase in absorbance, correlating to more Pi production (Fig. 2., and sample calculations).
A third ATPase assay, Assay C, contained the same treatments as assay A with decreasing concentrations of DCCD added to tubes C1-6, 10 mM- 0.625 mM, respectively. The absorbance values for tubes C1-6 were read at 660 nm. The highest absorbance reading (0.117) was found in the absence of DCCD in tube C6, while the lowest reading (0.027) correlated to the highest concentration of DCCD at 10mM. A steady decrease in Pi generated per 10 min was observed as [DCCD] increased during this treatment (Fig. 3.).
A final assay pertaining to the oxygen consumption, or ETC activity, was demonstrated with respiratory particles extracted from E. coli membranes using a French press to create inside-out vesicles. The values from the oxygen electrode with an attached pH meter were read at 2 minute intervals for 20 minutes. O2 levels were found to remain fairly constant around 8-10 O2 ppm in treatments 1, 2, and 5 containing buffer, membranes, and NADH + KCN, respectively. However, a sharp decline in O2 concentration was noted in treatments 3, containing NADH, and 4, containing NADH + Malonate, where the final concentration was close, or at, 0.0 O2 ppm (Fig. 4., and sample calculations).
Sample Calculations (Tube A4 at 10min)
-Amount of Pi Generated:
Pi = X µmoles
Ratio of VolumeATPase Assay: VolumeATPase Assay in Pi Assay x (X µmoles)
= [2.005 ml/0.250 ml(X µmoles)]
Pi in ATPase assay: 8.02(0.100 µmoles) = 0.802 µmoles
-ATPase Activity for 0.01 ml Membranes:
[8.02(0.100 µmoles)]/10 min = [0.802(0.100 µmoles/min)]/ 0.01 ml = 8.02 µmoles min-1 ml-1
-ATPase Specific Activity in Membranes:
(8.02 µmoles min-1 ml-1)/(10 mg/ml) = 0.802 µmoles min-1 mg-1
Sample Calculations for ETC Activity (NADH for 10 min)
-ETC Activity/ O2 Comsumption per ml:
ΔO2 for 8 min = 9.8 – 0.5 = 9.3 ppm
Volume of assay = 20 ml
[20(9.3)]/ [(16 µmole)(10 min)] = 1.11625 µmole min-1
-Specific ETC Activity/ O2 Comsumption per mg of Membrane
(1.1625 µmole min-1)/2 mg = 0.581 µmole min-1 mg-1
P:O Ratio = Specific Activity of ATPase: Specific Activity of ETC = 0.802/0.581 = 1.4

Table 1. Treatments given to each sample during the oxygen consumption assay:
Sample Number
1
2
3
4
5
Buffer (ml)
6.7
6.7
6.7
6.7
6.7
Water (ml)
13.3
13.1
11.8
10.6
11.1
Membranes (ml)
-
0.2
0.2
0.2
0.2
15 mM NADH (ml)
-
-
1.25
1.25
1.25
0.3 M Malonate (ml)
-
-
-
1.25
-
0.15 M KCN (ml)
-
-
-
-
0.75
Total Volume (ml)
20
20
20
20
20
Discussion
During this experiment, several errors prevented the data collection for assays A, B and C, data was collected from an adjacent group. First, the instructions were not properly examined, resulting in the addition of TCA before ATP in several tubes from each assay. This was an enormous error as the reaction was halted before Pi production could begin. Secondly, the intervals at which absorbance values were read were not within the specified 2.5 minute time-frame, which would result in a higher amount of Pi present in each tube. Lastly, transferring 0.250 ml aliquots from the original tube to a micro-centrifuge tube, and back to the clean tubes provided pipetting errors that decreased the amounts of Pi present in each tube.
Several assays were conducted - four ATPase assays and one ETC assay- to observe the relationship between ATPase phosphorylation and ETC oxidation. During the first assay with Pi and ANSA/ acid molybdate reagents, a blue colour in the reaction mixture gradually increased in darkness from tubes 1-6, containing 0.0-0.5 ml of Pi, respectively. In addition, a higher absorbance reading was obtained as the amount of Pi increased per tube. Thus, the colour intensity, and absorbance values read at 660nm, provide a measure of the amount of Pi present in each tube. The values obtained from this assay were used to produce a standard curve (Fig. 1.) from which the amounts of Pi in assays A and B were calculated (Sample Calculations).
The calculated amounts of Pi in assays A and B (Fig. 2.) showed that assay B produced lower amounts of Pi in the absence of Mg2+, than assay A, that contained Mg2+, 0.337, and 0.802, respectively, at the 10 minute interval. This suggests that the Mg2+/Ca2+ ATP synthase is dependent upon Mg2+ binding to produce optimal levels of Pi, or the reverse, ATP.
Values collected for the oxygen consumption assay showed that the sample containing NADH consumed the most O2, as indicated by the largest decrease (9.8 ppm at T=0, to 0.2 ppm at T=10), while the sample with KCN consumed the least amount of O2 with the smallest decrease in O2 concentration (remaining steady at 8.65 ppm). In addition, the sample given malonate also showed a sharp decline in O2 concentration with 9.1 ppm at T=0, to 1.1 ppm at T=10. This suggests that malonate and NADH are excellent substrates for the ETC as more oxygen was consumed during a 10 minute span than any other samples, with NADH O2 consumption slightly higher. This correlates with a higher activity of the ETC as electrons are removed from NADH and Malonate, and subsequently passed down the ETC to pump H+ ions out of the cytoplasm. The sharp depletion of O2 in both treatments suggests that O2 is quickly reduced to H2O during this process. In addition, the treatments containing buffer, membranes, and NADH + KCN maintained relatively constant oxygen concentrations throughout the assay. The treatment containing buffer and membranes should not have any activity as the ETC is not present, or provided with a substrate to oxidize. In the NADH + KCN sample, a minimal decrease in O2 concentration suggests that it has inhibitory effects on the activity of the ETC, as substrate was provided (NADH), but was not utilized. Thus, optimal ETC activity can be achieved with NADH and inhibited with the addition of KCN. While the literature values for the P:O ratio are 2, the data collected here yields a ratio of 1.4, resulting in an experimental error value of 30%. This could be due to experimental error such as incorrect addition of reagents, to pipetting errors, or possibly miscalculations.
Respiration can be classified as aerobic, or anaerobic, and occur by two different mechanisms; oxidative phosphorylation, and substrate-level phosphorylation. The former requires an ETC to produce an ion gradient that is used to turn the ATPase to form ATP. In aerobic respiration, the final electron acceptor is oxygen, while in anaerobic respiration, nitrate, sulphate, or other organic compounds are the final electron acceptors. While fermentation can occur with, or without the presence of oxygen –occurs more anaerobically-, it differs from oxidative phosphorylation in that the ETC is absent. There is no oxidative phosphorylation coupled between the ETC and ATPase. Instead, substrates, such as glucose, are directly oxidized to produce a lower amount of ATP.
In conclusion, optimal ATPase activity can be achieved with the addition of Mg2+ while optimal ETC activity can be achieved with addition of NADH. The importance of both complexes at maximal operating capacity is a higher output of ATP production for cellular energy.