Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Interaction of AMP-aminohydrolase with myosin and its subfragments.

We have shown that purified rabbit skeletal muscle AMP-aminohydrolase binds to rabbit muscle myosin, heavy meromyosin, and Subfragment 2 but does not bind to light meromyosin nor to Subfragment 1. The dissociation constant for binding to myosin was determined to be 0.14 muM. A new sedimentation boundary, presumably reflecting formation of a complex between AMP-aminohydrolase and heavy meromyosin or Subfragment 2, can be observed using the analytical ultracentrifuge. Binding of AMP-aminohydrolase to myosin, heavy meromyosin, or Subfragment 2 is abolished by phosphate (less than 10 mM), an inhibitor of AMP-aminohydrolase. No other rabbit muscle enzyme tested showed any interaction with myosin under the same conditions and there was no indication of complex formation between AMP-aminohydrolase and phosphofructokinase or phosphocreatine kinase in the analytical ultracentrifuge.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

The reversibility of adenosine triphosphate cleavage by myosin

For the simplest kinetic model the reverse rate constants (k₋₁ and k₋₂) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl₂, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k₋₁<0.02s⁻¹ and k₋₁=16s⁻¹. Apparently, higher values of k₋₁ and k₋₂ are found with less-purified protein preparations. The values of k₋₁ and k₋₂ satisfy conditions required by previous ¹⁸O-incorporation studies of H₂¹⁸O into the P_i moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.