Die erschöpfende reinigung von aktin-präparaten zahl und art der phosphathaltigen prosthetischen gruppen von G- und F-aktin

Abstract

Exhaustive purification of actin preparations. Number and kind of phosphate containing prosthetic groups of G- and F-actin 1. 1. Previous investigations on the nucleoside phosphate content of G- and F-actin have all been carried out with unpurified or little purified protein preparations. It has never been tested whether the purification was complete or whether the purification itself inactivated the preparation. 2. 2. In F-actin solutions prepared according to Straub the content of adenine decreases by repeated ultracentrifugal sedimentation or Mg-precipitation according to Bárány to a constant level which is identical with both methods (16 μmoles/g protein). The adenine and phosphate content of the actin remains constant after the second purification procedure. Both procedures remove protein impurities present in the crude extract. 3. 3. The protein impurities are, however, not removed by repeated isoelectric precipitation of the crude unpolymerized Straub-extract. This procedure removes only contaminating phosphate and nucleoside phosphate of the crude extract. The actin polymerizes spontaneously during isoelectric precipitation. 4. 4. Ultracentrifugal sedimentation of F-actin, precipitation by MgCl 2 or isolectric precipitation in presence of ATP do not inactivate the actin. The viscosity of F-actin, the ability for activating the ATP-ase of added L-myosin and the ATP-sensitivity of the resulting actomyosin remain constant even after repeating the purification procedure five times. 5. 5. Repeated isoelectric precipitation of actin in absence of ATP leads to an increasing loss of adenine phosphate and also to a stepwise decrease of Zν and ATP-sensitivity. 6. 6. In the nucleoside phosphate of purified F-actin the proportion of adenine to phosphate is 1:2 as in ADP. Paperchromatographic methods reveal in addition traces of AMP and ATP. 7. 7. G-actin and the contaminating proteins in the crude extracts contain also 16 μmoles adenine/g protein. 8. 8. From the content of adenosine phosphates bound to G- or F-actin (16 μmoles/g protein) the minimal molecular weight of the actin monomer is calculated as 62.000. 9. 9. The proportion adenine: phosphate and paperchromatographic methods show, that in the crude unpolymerized extract the protein-bound nucleoside-phosphate consists of 70–75% ATP and 25–30% ADP. Only 70–75% of the protein in the crude extract are able to polymerize. 10. 10. However, G-actin obtained from purified F-actin containing also 70–75% of its nucleoside phosphates as ATP does polymerize entirely. Thus whether or not ADP-G-actin polymerizes seems to depend on the history of the protein preparation. 11. 11. G-actin, whose ability for polymerization has been destroyed by X-rays, nevertheless activates the L-myosin-ATP-ase to a normal extent. The same holds for actin partly denatured by isoelectric precipitation in the absence of ATP. Thus, the ability of actin for polymerisation and its ability to activate the L-myosin-ATP-ase are independent properties. 12. 12. In phosphate or ATP containing solutions, purified F-actin ATP and inorganic phosphate reversibly (in addition to the tightly bound ADP). Howeever, actin is not phosphorylated in presence of ATP.