AMP-PNP is the abbreviation for a structural analogue of ATP in which the second

Question

AMP-PNP is the abbreviation for a structural analogue of ATP in which the second and third phosphate groups are linked by an NH group (instead of an oxygen atom). AMP-PNP binds to the ATP-binding site of most ATPases. It differs from ATP in that the terminal phosphate cannot be removed by hydrolysis (so you cannot create ADP). Explain the following a) When isolated myofibrils are placed in a flask containing Ca2+ and AMP-PNP, contraction is quickly arrested. Where in the contraction cycle will contraction stop? Be specific. Draw two individual sarcromeres to show what is happening (draw a sarcomere before the cells are put in the flask and draw one after in has been in the flask). b) What would happen to a sperm flagellum if which AMP-PNP is added? Be specific as to the proteins/molecules whose function would be inhibited and what would be the overall effect? c) When researchers incubated purified vesicles, axoplasm, and MT from the giant squid axon and added AMP-PNP, the vesicles no longer moved. Explain this observation including specific proteins/processes affected (think about what proteins move vesicles). Diagram your explanation. d) Provide your own example of cell motility where AMP-PNP would affect movement.

Explanation / Answer

1. AMP-PNP (Adenylylimidodiphosphate) a nonhydrolysable analogue of ATP is used to arrest the crossbridge cycle of muscular contraction. X-ray diffraction is used to study the effect of binding of AMP-PNP on the structure and spatial arrangement of cross-bridges.

When there is absence of enough ATP there is no dissociation of cross-bridge which causes the muscle to become stiff and locked. Therefore a rigid state is formed known as rigor. Rigor mortis is common phenomenon after death in which there is stiffening of dead body due to accumulation of cross bridges due to depletion of ATP.

In cross- bridge cycle actin (A) combines with myosin (M) and ATP is converted to ATP and Pi.

The AMP-PNP cannot be hydrolysed to ADP and Pi therefore though the cross bridges dissociate from the thin filaments it cannot take high energy conformation because AMPPNP cannot hydrolyze to ADP and Pi.

The arrested myofibrils can resume its structure and function if enough ATP is added so that the affinity for ATP is higher as compared to AMP-PNP.

2. When AMP-PNP is added to the sperm flagellum it will arrest the movement of flagellum by tightly binding to the microtubules. This led to isolation of motor protein kinesin which remain bound to the microtubules in presence of AMP-PNP but is released in presence of ATP.

Like the dynein and myosin the kinesin is also an elongated protein containing two heads and a tail. The movement of dynein and kinesis along a microtubule is the same like the sliding movement of myosin along an actin microfilament.

3. When the extract is provided with ATP the movement of the vesicles can be seen along the microtubules through video microscopy. The rate of this movement is approx. 1-2 µm / s. This movement in the cell free system is similar to the movement in the intact cell.

When such extract is provided with AMP-PNP the vesicles bound tightly to the microtubules and did not move. This suggests that movement requires the hydrolysis of terminal phosphate bond in ATP.

The protein responsible for the arresting the movement of microtubule is Kinesin. Kinesin isolated from squid axoplasm is a dimer of two heavy chains. The heavy chains forms complex with light chain. The molecule has three domains with three different functions :- the head domain binds microtubules and ATP and is responsible for the motor activity and the tail domain which is responsible for binding with the membrane bound vesicles. The term ‘Kinesin cargo’ is given to the membrane bound vesicle.

4. An example of cell motility affected by AMP-PNP is the chromosomal movements by the action of spindle microtubules. Cin8p a kinesin related mitotic spindle motor protein from S. cerevisae its binding to microtubules is very important for motility. The yeast cell overproducing Cin8p showed bundling activity. In presence of AMP-PNP the microtubular structures were observed as bundles. When its concentration was reduced bundling was reduced.

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