Glycogen, TCA Cycle and Mitochondria (Lecture 13)

Question 1

evidence for chemo-osmotic coupling: (6)

Answer 1

the respiratory chain can function in the absence of phosphate

the # moles of ATP generated through NADH oxidation was a not an integer

an intact IMM is required for OXPHOS

key e- transport proteins pan the IMM

uncouplers such as 2,4-Dinitrophenol (DNP) inhibits ATP synthesis

generate an artificial proton gradient permits ATP synthesis with out e- transport

Question 2

what does DNP do?

Answer 2

weak acid that takes H+ form the IMM and can release H+ back inept the matrix. this decreased the proton motor force

respiration can still occur, as for consumption of oxygen can occur (even if ATP synthesis is impaired)

Question 3

how did they test the proton gradient

Answer 3

artificial proton gradient in chloroplasts (opposite to mitochondria, plump in) when chloroplasts are incubated, they have many positive charges within the organelle, thus making that environment acidic.

when placed in a buffer of a larger pH , and adding ADP + Pi, ATP can be synthesized with simply an electrochemical gradient

permeabilizing the membrane removed the electrochemical gradient, thus no ATP synthesized

Question 4

what does beta hydroxybutyrate do?

Answer 4

NADH linked respiration will be generated.

go into CAC and NADH is generated, thus e- can enter complex I

Question 5

what occurs when rotenone/amytal is added?

Answer 5

inhibits NAD+ linked oxidation.

this inhibits complex I form passing its e-

Question 6

what occurs when succinate is added?

Answer 6

adding e- via complex II to bypass complex I and allow e- to pass into CoQ

FAD linked oxidation occurs

Question 7

what occurs when antimycin is added?

Answer 7

inhibits FAD-linked oxidation, this blocks any e- upstream of complex III (blocks complex III)

Question 8

what occurs when Ascorbate or TMPD is added?

Answer 8

electrons are donated to cytochrome C to complex IV via the artificial electron donor

oxidation resumed

Question 9

what occurs when CN (cyanide) is added?

Answer 9

oxidation terminates

this blocks complex IV

Question 10

how did they identify how complex I contributed ?

Answer 10

inhibited complex III with antimycin

added ferricyanide was added as an artificial e- acceptor so that e- could continue to flow if they were donated upstream

this allows e- to com e into complex I and be released into ferricyanide, so that they sent have to travel down the closed complex III

this allows complex I to pump protons

the P/O ratio was 1, 1ATP was generated

Question 11

how did they identify how complex IV contributed ?

Answer 11

using artificial e- donors, TMPD and ascorbate will give e- into cytochrome c

P/O ratio = 1ATP

Question 12

how did they identify how complex III contributed ?

Answer 12

added exogenous cytochrome C to allow e- to flow (be accepted). this was done because complex IV was blocked with CN. this forced e- to exit via th exogenous cytochrome c

complex I was bypassed via succinate, inhibited complex IV and the e- flowed through the exogenous cytochrome c

P/O =0.5

this was not complex II’s ATPS but this activity of was inhibited but antimycin, a complex II inhibitor

Question 13

DNP

Answer 13

high amount of protons relative to the matrix, it can pick up these protons (lipophilic , thus goes through mitochondrial membrane )

it will release the proton to the matrix, ewe there is a lower concentration of protons

this is a proton leak pathway, thus decreasing the proton motor force/ electrochemical gradient , this is a stimulus to consumes and oxidize more NADH and pump more protons to maintain the PMF

causes weight loss, generating heat rather than storing energy

UPC1 is an endogenous uncoupler (might combat obesity)

Question 14

mitochondrial origin

Answer 14

archer engulfed bacteria, which provided the host with oxidizable substrates and provided the endosymbiont with energy

most of the genes of the endosymbiont were transferred to the host ( within the nuclear genome)

Question 15

genes encoding the protein in the ETC are from

Answer 15

genes encoded in mitochondrial DNA and nuclear DNA

Question 16

which substrates are needed in adequate amounts to proceed with oxidative phosphorylation?

Answer 16

Pi

ADP

O2

oxidizable metabolites that can generate reduced e- carrier (NADDH, FADH2)

Question 17

Respiratory control

Answer 17

electron flow though the ETC to make ATP via ATP synthase, but e- flow only occurs when ATP is ending synthesized

if ATP builds yup, the e- flow will stop

energy generating capacity of the cells is determined by energy demands

Question 18

experiments to determine respiratory control

Answer 18

adding glutamate, recall that glutamate is converted to alpha KG via the transamination reaction

the regeneration of of of CAC intermediates drives respiration

adding ADP respiration increases until it comes back to baseline levels. this baseline is met when ADP is depleted to normal levels, thus enough ATP is produced

when adding more ADP, oxygen consumption was reinitiated, thus indicating that it was limiting

Question 19

if after adding ADP, the respiration levels remain at baseline, what may explain this

Answer 19

consumed all of the oxygen (O2 limiting)

low CAC flux, because perhaps glutamate concentrations were limiting

Question 20

what does oligomycin do?

Answer 20

blocks ATP synthase because no ATP is produced and when DNP is added (uncoupler, which increases respiration independently of ATP synthesis) e- flow proceed to increase respiration (but not ATP generated)