Purifica=
tion
of smooth muscle myosin1
Smooth muscle myosin is
prepared from frozen turkey gizzards. Minced muscle is extensively
extracted at low ionic strength with detergent to remove membrane and solub=
le
proteins leaving myofibrils. Myosin is extracted by dissociation with ATP in
the absence of magnesium or calcium ions. Actin=
and
associated proteins are precipitated with 150 mM MgCl2
while ATP dissociated myosin remains soluble. Myosin is precipitated by
dilution to 15 mM MgCl2. Collected myosin is dissolved in h=
igh KCl in which contaminating actin=
remains insoluble. Finally, m=
yosin
is precipitated with 10 mM MgCl2,
collected and dissolved in high KCl.
= 1. Thaw turkey gizzards and trim them of fat and connective tissue and mince.
= 2. Suspend 100 gm of mince in 3 volumes of Buffer A (10 mM= Tris-HCl , pH=3D7.5, 50 mM KCl, 2 mM ethylenediaminetetracaetic acid (EDTA), 25 mM MgCl2, 0.2 mMDTT, 3% vol/vol Triton = X-100) at 4o and homogenize for 20 seconds in a chilled Waring blendor in a cold room.
= 3. Centrifuge the homogenate at 3,000 rpm in a Sorvall GS-3 r= otor for 5 minutes at 4o.
= 4. Discard the supernatant. Homogenize and centrifuge the pellet 3 more times as above= .
= 5. Suspend the pellet in 3 volumes of Buffer B (10 mM Tris-HCl, pH=3D7.5, 100 mM KCl, 2 mM EGTA, 0.2 mMDTT) and homogenize for 20 seconds.
= 6. Centrifuge the homogenate at 7,000 rpm in a Sorvall GS-3 r= otor for 10 minutes at 4o.
= 7. Discard the supernatant. Homogenize and centrifuge the pellet two more times as abo= ve. This produces a white residue of myofibrils.
= 8. Suspend the pellet in 1.5 volumes (based on the original mince weight) of Extraction Buffer (40 mM imidazole, pH=3D6.8, 5 mM ATP, 4 mM EDTA, 2 mM EGTA, 0.5 mMDTT= ) and homogenize for 20 seconds.
= 9. Adjust the pH to 6.9 and leave on ice for 15 minutes with occasional stirring. This step extracts myosin.
= 10. Centrifuge the mixture at 11,000 rpm in a Sorvall SS-34 ro= tor for 20 minutes at 4o.
= 11. Discard the pellet. Filter the supernatant through glass wool in a funnel.
= 12. Adjust the pH of the filtrate to 7.6 with 0.1 N KOH and measure the volume.
= 13. Slowly add 1 M MgCl2 with stirring to a final concentration of 150 mM (0.177 volume of Step 12) using a peristaltic pump= .
= 14. Add 100 mM ATP (neutralized) to 2.5 mM (0.03 volume of 12) and stir gently for 10 minut= es on ice. = = &nb= sp;
= 1. Important Note: The addition of M= g ions to phosphate and ATP and the hydrolysis of ATP by myosin result = in considerable liberation of protons. Therefore he= pH should be monitored and maintained at 7.6 with 0.1 N KOH through Steps 13 a= nd 14.
= 15. Centrifuge at 11,000 rpm in a Sorvall SS-34 rotor for 20 m= inutes at 4o.
= 16. Centrifuge the supernatant at 30,000 rpm in a Beckman Type 45 Ti rotor overnight at 4<= sup>o.
= 17. Measure the volume of the supernatant. Slowly dilute with 10 volumes of 4o distilled water while stirring. The solution should become turbid from precipitated myosin.
= 18. Centrifuge at 8,000 rpm in a Sorvall GS-3 rotor for 10 min= utes at 4o.
= 19. Using a large-bore pipette, suspend the myosin pellet in 10 pellet volumes of Buf= fer C (10 mM sodium phosphate, pH 7.6, 0.5 mM EGTA. 5 mM ATP) and ho= mogenize in a teflon-glass homogenizer until a homogeneo= us opaque suspension is produced.
= 20. Adjust the pH to 7.6 with 0.1 N KOH. Measure the volume.
= 21. Slowly add 1 M MgCl2 to a final concentration of 150 mM (0.177 volume) with stirring. Monitor the pH as = above.
= 22. Centrifuge at 40,000 rpm in a Beckman Type 45 Ti rotor for 3 hours at 4o.
= 23. Measure the supernatant and dilute it with 10 volumes of 4o distilled water while stirring.<= /span>
= 24. Centrifuge at 10,000 rpm in a Sorvall SS-34 rotor for 10 minu= tes at 4o to collect myosin.
= 25. Dissolve the myosin pellet in 10 pellet volumes of Buffer D (10 mM Tris-HCl, pH 7.5, 0.5 M KC= l, 1 mMDTT) using a teflon-glass homogenizer.
= 26. Measure volume. Dilute with 4 volumes of 4o distilled water while stirri= ng to give a final KCl concentration of 0.1 M.
= 27. After 15 minutes on ice, centrifuge at 8,000 rpm in a Sorval= l SS-34 rotor for 15 minutes at 4o. The supernatant is myosin. The pellet contains myosin plus contaminant actin. = The size of the pellet varies, but a large pellet representing more than 30% of total myosin (compare to pellet from Step 24) indicates a failure to proper= ly maintain pH during earlier MgCl2 precipitation steps.
= 28. Measure supernatant volume. Dilute the supernatant with an equal volume of 0o<= /sup> distilled water while stirring. Add 1 M MgCl2 dropwise to 10 mM (1/100 volume).
= 29. Place on ice= for one hour and centrifuge at 10,000 rpm in a Sorvall SS-34 rotor for 15 minutes at 4o to pellet myosin.
= 30. Dissolve the pellet with about 10 ml of Buffer E=3D 10 mM N-(-2-hydroxyethyl)piperazine-N-(4-butanesulfonic acid (HEPES), pH=3D 7.6, 0.5 M KCl, , 1 mM DTT and dialyze versus 500 ml of this buffer overnight at= 4o.
= 31. Centrifuge in a Beckman 45 Ti rotor at 40,000 rpm (k=3D170) for 1 hr at 4o.=
= 32. Dialyze against 500 ml Buffer F: 10 mM HEPES, pH=3D 7.5= , 20 mM KCl, 2 mM MgCl2, 1 mM DTT overnight. The solution will = become turbid due to myosin assembly. Drop freeze aliquots in liquid nitrogen.
Note: Alternatively, myosin from Step 31 can be precipitated with 50% ammonium sulfate and stored at 4o for several months.
The typical yield of myosin is 2.5 milligrams/gm of gizzard mince. The extinction coefficient for myosin at 280 nm is 0.56 ml milligram-1cm-1= .
Solutions:
Note: Make all buffers fresh and store at 4o.
Buffer A: 1.5 l=3D Tris-base 1.82 g, KCL 5.59 g, EGTA 1.14 g,= MgCl2 7.62 g, DTT 0.046 g, Triton X-100 45 ml; titrate to pH 7.5.
Buffer B: 1.0 l=3D Tris-base 1.21 g, KCl= 7.46 g, EGTA 0.76, DTT 0.031 g; titrate to pH 7.5.
Extraction Buffer: 200 ml=3D&= nbsp; imidazole 0.55 g, Na2A= TP 0.55 g, Na2EDTA 0.27 g, EGTA 0.15 g, DTT 0.015 g; titrate to pH = 6.8.
100 mM Na2ATP= trihydrate: 10 ml=3D 0.60 g; neutralize. Store frozen= at -20o.
1 M MgCl2 hexahydrat= e: 100 ml=3D 20.3 g
Buffer C: 100 ml=3D Na2HPO4 0.14 g, EGTA 0.019 g, Na2ATP= 0.28 g; titrate to pH 7.6.
Buffer D: 100 ml=3D Tris-base 0.12 g, KC= l 3.73 g, DTT 0.015 g; titrate to pH 7.5.
Buffer E: 1.0 l=3D Na HEPES 2.60 g, KCl 37.3 g, DTT 0.15 g; t= itrate to pH 7.6.
Buffer F: 500 ml=3D Na HEPES 1.30 g, KCL 0.75 g, MgCl2 0.20 g, DTT 0.077 g; ti= trate to pH 7.5.
100% ammonium sulfate: 100 ml=3D 76.7 g
1 This proto= col is a modification of M. Ikebe and D.J. Hartshorne, J. Biol. Chem. 260: 14146-13153 (1985).