When vesicles of the cardiac sarcoplasmic reticulum (CSR) are incubated with ATP
ID: 3474480 • Letter: W
Question
When vesicles of the cardiac sarcoplasmic reticulum (CSR) are incubated with ATP Mg^2+ and Ca^2+, they take up Ca^2+ and reach a pseudo steady-state. This is a steady state that changes, but slowly. The uptake of Ca2+ is mediated by the SERCA2a Ca-ATPase. The uptake reaction can be quenched by adding EGTA to the external solution, which binds the Ca^2+ outside of the vesicles, or by adding glucose plus hexokinase, that converts the ATP to ADP and glucose-6 phosphate. When the uptake reaction is stopped, Ca^2+ that was already taken up by the vesicles leaks out passively A. The amount of Ca^2+ taken up by the vesicles is generally normalized to the amount of CSR protein in mg rather than being expressed as a concentration. A typical steady state Ca2 uptake is 40 nmol mg^-1. In separate experiments, the enclosed volume of the CSR vesicles was determined to be 5 mu L mg. what is the approximate concentration of Ca^2+ inside the vesicles at steady-state? The total Ca^2+ concentration is its amount divided by the volume in which it is distributed. In this case the amount is 40 nmal mg CSR protein^-1 and the volume is 5 mu L mg CSR protein^-1, The concentration is estimated as B. The average vesicle size determined by electron microscopy is about 150 nm. What is the volume and surface area of a vesicle this size, assuming it is a sphere? C. Given that the enclosed volume of the aggregate vesicles is 5 iL mg-1, how many vesicles are there per mg of CSR protein? How much surface area is there per mg of CSR protein? D. The initial passive efflux at a load of 40 nmol mg when the pump is stopped is 16 nmol min^-1mg^-1. Convert this to a flux in units of nmol cm^-2 s^-1 by dividing by the surface area per mg of CSR protein and converting min to s. E. What is the passive permeability to Ca^2+ in cm s^-1?Explanation / Answer
A. Amount of Ca2+ taken by vesicle at steady state= 40nmol/ mg
The enclosed volume of CSR vesicle = 5uL/mg
Concentration of Ca2+ inside the vesicle = 40 x 10-9 mol/ 5 x 10-6 L = 8 x 10-3 mol/ L (equivalent to 8000nmol/ cm3)
B. Volume and surface area of the spherical vesicle of size 150nm; Radius of the vesicle = 75nm
Volume of the spherical vesicle = 4/ 3 x (pi) x r3 = 4/3 x 22/7 x (75 x 10-9 )3 = 1.77 x 10-21 m3
= 1.77 x 10-18 L (since 1m3 is equivalent to 1000L)
= 1.77 x 10-12 uL
Surface area of the spherical vesicle = 4 x (pi) x r2 = 4 x 22/7 x (75 x 10-9)2 = 7.06 x 10-14 m2(equivalent to 7.06 x 10-10cm2 ).
C. No. of spherical vesicles/ mg of CSR proteins = 5 x 10-6 / 1.77 x 10-18 = 2.82 x 1012
Total surface area = 2.82 x 1012 x 7.06 x 10-10cm2 = 19.94 x 102 cm2 / mg of CSR proteins
D. Convert the flux 16 nmol min-1mg-1to nmol cm-2 sec-1 = 16 / (19.94 x 102 x 60) = 1.34 x 10-4 nmol cm-2 sec-1
E. Passive permeability = Flux/ concentration of Ca2+ (in terms of nmol/ cm3 , pl see answer a.)
= 1.34 x 10-4nmol cm-2 sec-1/ 8000nmol cm-3=1.675 x 10-8 cm sec-1
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