Practical E3

Question 1

Uncouplers are …

Answer 1

weak acids that transport the protons back into the matrix directly, bypassing the ATP synthetase

Question 2

2,4-dinitrophenol

Answer 2

the classic example of an uncoupler that is is water-soluble when deprotonated, but lipid soluble when protonated, so that it will diffuse across the crista membrane down its concentration gradient.

Question 3

site A - site B - site C-

Answer 3

ADP + Pi enter ATP created, H20 leave, proton used ATP leave

Question 4

At any given time, each site is at a different stage in the reaction:

Answer 4

• One site is binding ADP and phosphate • One site is catalysing the condensation of ADP and phosphate, and expelling and water • One site is expelling ATP, ready to accept ADP and phosphate

Question 5

ATP synthetase - describe

Answer 5

As protons enter through the stalk of the primary particle that spans the crista membrane, so they cause a rotation of the central part of the ATP synthetase, forcing each site in turn to proceed to the next step in the reaction.

Question 6

Electron transport and substrate oxidation are controlled by the availability of ADP

Answer 6

If there is no ADP available to bind to the empty site, then rotation of the central part of the ATP synthetase is not possible, and protons cannot cross the stalk of the primary particle. This leads to an accumulation of protons in the crista space, which, in turn, inhibits any further expulsion of protons by the electron transport chain, so that electron transport (and therefore substrate oxidation) ceases.

Question 7

A wide variety of different compounds act as respiratory poisons, and inhibit the oxidation of metabolic fuels linked to the phosphorylation of ADP to ATP.

Answer 7

• Cyanide and carbon monoxide, well known poisons. • Sodium azide - another well-known poison • Antimycin - one of a family of antibiotics produced by Streptomyces spp., some of which are used as fungicides against fungi that are parasitic on rice. • Amytal (amobarbital) - a barbituric acid derivative widely used as a sedative or hypnotic drug. • Rotenone - the main insecticidal compound in derris powder, extracted from the root of the leguminous plantLonchocarpus nicou. • Oligomycin - one of a family of antibiotics produced by Streptomyces spp., which is of little or no therapeutic use. • Atractyloside - a toxic alkaloid from the rhizomes of the Mediterranean thistle Atractylis gummiferra. It competes with ADP for binding to the adenine nucleotide transporter. • Bongkrekic acid - a toxic antibiotic formed by Pseudomonas cocovenans growing on coconut. It anchors the adenine nucleotide transporter at the inner face of the membrane, so that ATP cannot be transported out, nor ADP in.

Question 8

Consumption of dinitrophenol leads to ….

Answer 8

an increased metabolic rate, high oxygen consumption and a high body temperature. As you perform experiments with dinitrophenol added to your mitochondrial preparation you should be able to deduce how its acts to stimulate substrate oxidation.

Question 9

Calculate PO ratio for a given graph:

Answer 9

You should draw straight lines, as shown above, to determine the % oxygen saturation:
A) at the time that the mitochondrial preparation is added
B) at the time that ADP is exhausted

Then measure the distance from 0 - 100% oxygen saturation

In this example, by measurement (on the original printed graph)t:
100% oxygen saturation (1327 nmol O) = 78 mm

point A = 72.5 mm = 72.5 / 78 = 0.929 x 1327 nmol O = 1233 nmol O

point B = 40 mm = 40 / 78 = 0.512 x 1327 nmol O = 680 nmol O

Hence, difference = 1233 - 680 = 552 nmol O

You should be able to calculate the P:O ratio from this value (the amount of oxygen used) and the amount of ADP you added in the experiment.