Glycogen, TCA Cycle and Mitochondria (Lecture 13) Flashcards
evidence for chemo-osmotic coupling: (6)
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
what does DNP do?
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)
how did they test the proton gradient
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
what does beta hydroxybutyrate do?
NADH linked respiration will be generated.
go into CAC and NADH is generated, thus e- can enter complex I
what occurs when rotenone/amytal is added?
inhibits NAD+ linked oxidation.
this inhibits complex I form passing its e-
what occurs when succinate is added?
adding e- via complex II to bypass complex I and allow e- to pass into CoQ
FAD linked oxidation occurs
what occurs when antimycin is added?
inhibits FAD-linked oxidation, this blocks any e- upstream of complex III (blocks complex III)
what occurs when Ascorbate or TMPD is added?
electrons are donated to cytochrome C to complex IV via the artificial electron donor
oxidation resumed
what occurs when CN (cyanide) is added?
oxidation terminates
this blocks complex IV
how did they identify how complex I contributed ?
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
how did they identify how complex IV contributed ?
using artificial e- donors, TMPD and ascorbate will give e- into cytochrome c
P/O ratio = 1ATP
how did they identify how complex III contributed ?
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
DNP
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)
mitochondrial origin
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)
genes encoding the protein in the ETC are from
genes encoded in mitochondrial DNA and nuclear DNA
which substrates are needed in adequate amounts to proceed with oxidative phosphorylation?
Pi
ADP
O2
oxidizable metabolites that can generate reduced e- carrier (NADDH, FADH2)
Respiratory control
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
experiments to determine respiratory control
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
if after adding ADP, the respiration levels remain at baseline, what may explain this
consumed all of the oxygen (O2 limiting)
low CAC flux, because perhaps glutamate concentrations were limiting
what does oligomycin do?
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)
