By Sidney Fleischer, Becca Fleischer
The shipping volumes of the Biomembranes sequence have been initiated with Volumes a hundred twenty five and 126 of tools in Enzymology. those volumes coated delivery in micro organism, Mitochondria, and Chloroplasts. Volumes 156 and 157 hide ATP-Driven Pumps and comparable Transport.
The subject of organic membrane delivery is a really well timed one simply because a powerful conceptual foundation for its knowing now exists
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Biomembranes Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps
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Extra resources for Biomembranes Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps
Example text
Temperatures of 00-5 ° for amphibian muscles and 200-25 ° for mammalian and avian muscles are recommended. Higher temperatures can be used, but they tend to cause a very rapid rundown of skinned fibers. Protocols for the measurement of Ca2? release in skinned fibers essentially consist of three steps. Starting from the empty SR, (I) load the SR with Ca 2÷ to a fixed level by immersing the fiber in a medium containing Ca 2÷ and MgATP for a period of time, and allow the SR to accumulate Ca z÷ by Ca2+-pump ATPase.
The uptake reaction is started by addition of concentrated Tris-ATP to a final concentration of 5 mM. ~m Millipore filters. 5 mM NaCI. The arrow indicates the addition of 1 /xM monensin, a Na + ionophore, which releases the sodium accumulated. 3, and a protein concentration of 40/~g/ml. The sample is then further preincubated for 30 min at 37°. 2), which initiates the uptake reaction. 22-/xm Millipore filter disks (GSWP) (Bedford, MA). The filters are immediately washed three times (2 ml/wash) with wash medium.
If it is too low, Ca 2÷ loading may already start in the relaxing solution because of contaminating Ca 2+. Ca 2÷ loading (Step 1) starts by applying the loading solution (typical composition is given in Table I). A Ca 2+ concentration that does not directly activate the contractile system is usually preferred. The Ca 2+ concentration should be buffered with a high concentration of a Ca 2÷buffer. After an appropriate period of time, the loading solution is replaced by a relaxing solution having a high concentration of EGTA to stop the loading immediately.