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Lecture 8

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Biology 1002B
Tom Haffie

1. Change in respiration rate (oxygen consumption) in isolated mitochondria by NADH, ADP, uncoupler • Mitochondria is added. ◦ Initially there is no oxygen in the chamber (mitochondria) ◦ But when you add mitochondria, the rate of oxygen (the slope) is going downward- less steep. That means that oxygen is being consumed in the chamber. • NADH is added ◦ the slope is decreasing- steeper downward- when NADH is added. The rate of oxygen consumption is going up. ◦ It is because you have isolated mitochondria. Isolated mitochondria doesn't have the source of substrate. They're disconnected from glycolysis. There is no pyruvate being transported into the mitochondria. So you're gonna quickly deplete (diminish in quantity) the level of NADH within the mitochondria matrix. ◦ Then when we add NADH, we add substrate, the rate of Electron Transport – oxygen consumption goes up. • ADP is added ◦ it would go even faster. ◦ If we add ADP, it would affect ATP synthesis since ADP+Pi -> ATP, however, Oxygen consumption in ETC is independent with ATP synthesis, therefore it would not affect the rate of oxygen consumption. But is it true? No!! they are coupled. Thus, they influence each other. ◦ But the rate will go even faster when we add ADP. This is related to respiratory control. ◦ If there is no ADP gradient around, then proton gradient in the intermembrane will become very high. pH in the inter membrane becomes low and it becomes harder and harder for these complexes to pump proton to the space that already has a lot of proton and that limits the rate of ET. The fact that you already have a huge proton gradient established, limit the overall rate of electron transport. These gradient is high , because you have no substrate to drive the phosphorylation of ATP. • Uncoupler is added. Added another substrate. - THE HIGHEST RATE ◦ ex: 2,4 Dinitrophenol ◦ the rate will go really really really fast. Steeper slope, the rate goes up. Uncoupler give a giant hole in the membrane. There is no (zero) proton gradient, no back pressure. It is so easy to pump proton across the membrane. No pH difference across the membrane. NOTE: when you add ADP, there is still pH difference across the membrane ◦ you lose your respiratory control, ET goes as fast as it possibly can. Proton being pump, but it is leaking right back into the matrix. ET and chemiosmosis are totally uncoupled. 2. Definition of respiratory control and how it is accomplished (proton gradient). • Respiratory control is ETC and oxygen consumption are regulated by the abundance of ADP, in this case. • The limitation placed on electron transport by the chemisosmotic gradient is termed respiratory control. Mitochondria are said to exercise respiratory control as long as they can restrict electron transport by means of the gradient. If the gradient is destroyed by damaging the membranes, respiratory control is abolished and electron transport can run freely. Uncouplers / Respiratory Control / P/O Ratio Uncouplers: bind protons, are hydrophobic and can dissipate a pH gradient by equilibrating H+ (protons). i.e. Dinitrophenol (DNP), causes ATP formation to cease but oxygen consumption remains rapid in an attempt by the mitochondria to maintain the proton gradient. Energy is released as heat and body temperature rises. Rapid O 2onsumption in uncoupling is due to loss of respiratory control. Respiratory Control: 1. Depends on ADP and P regilating O use.2
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