Comparative studies on the structure and aggregative properties of the myosin molecule. I. The structure of the lobster myosin molecule.

Myosin purified from the abdominal flexor muscle of the lobster, Homarus americanus, has a number average length of 1559 +/- 218 A, a rod like tail 1335 A long and a globular head 225 X 45 A as determined from electron microscopic observations on platinum shadowed preparations. The mass of the molecule was determined to be ca. 486,000 daltons from high speed equilibrium centrifugation studies at neutral and alkaline pH, and by SDS-acrylamide gel electrophoresis. Both sedimentation equilibrium centrifuge studies at alkaline pH and SDS-acrylamide gel electrophoresis experiments, indicate that the molecule contains a heavy chain core (two polypeptide chains weighing ca. 210,000 daltons each) and ca. four light chains of two weight classes (ca. 16,000 and 20,000 daltons). The amino acid composition of the myosin was determined. The specific activities of the Mg2+ -activated, K+/EDTA-activated, and Ca2+ -activated ATPases of the myosin were determined. Kinetic analysis of the digestion of lobster myosin with trypsin suggests that lobster myosin contains three classes of lysine and arginine residues; slowly split (k = 2.07 +/- 0.31 X 10(-2) moles/min2), rapidly split (k = 11.0 +/- 1.83 X 10(-2) moles/min2) and trypsin insensitive. There are 187 +/- 22 slowly split residues, 280 +/- 35 rapidly split residues, and 144 +/- 41 trypsin insensitive bonds per molecule. Comparison of these molecular parameters with those for the vertebrate skeletal muscle myosin indicates that the two myosins are similar in terms of mass, shape and overall polypeptide chain composition but may be considerably different in terms of local polypeptide chain conformation or composition.     

Human platelet myosin. II. In vitro assembly and structure of myosin filaments

We have used electron microscopy and solubility measurements to investigate the assembly and structure of purified human platelet myosin and myosin rod into filaments. In buffers with ionic strengths of less than 0.3 M, platelet myosin forms filaments which are remarkable for their small size, being only 320 nm long and 10-11 nm wide in the center of the bare zone. The dimensions of these filaments are not affected greatly by variation of the pH between 7 and 8, variation of the ionic strength between 0.05 and 0.2 M, the presence or absence of 1 mM Mg++ or ATP, or variation of the myosin concentration between 0.05 and 0.7 mg/ml. In 1 mM Ca++ and at pH 6.5 the filaments grow slightly larger. More than 90% of purified platelet myosin molecules assemble into filaments in 0.1 M KC1 at pH 7. Purified preparations of the tail fragment of platelet myosin also form filaments. These filaments are slightly larger than myosin filaments formed under the same conditions, indicating that the size of the myosin filaments may be influenced by some interaction between the head and tail portions of myosin molecules. Calculations based on the size and shape of the myosin filaments, the dimensions of the myosin molecule and analysis of the bare zone reveal that the synthetic platelet myosin filaments consists of 28 myosin molecules arranged in a bipolar array with the heads of two myosin molecules projecting from the backbone of the filament at 14-15 nm intervals. The heads appear to be loosely attached to the backbone by a flexible portion of the myosin tail. Given the concentration of myosin in platelets and the number of myosin molecules per filament, very few of these thin myosin filaments should be present in a thin section of a platelet, even if all of the myosin molecules are aggregated into filaments.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Segmental flexibility in the myosin molecule: Evidence from binding studies of myosin fragments with actin

From comparative studies of the association with polymeric actin of the bifunctional species heavy meromyosin and its monofunctional constituents, information about the relative freedom of these paired elements can be derived. An isotherm for the former binding process is presented which involves, as an experimentally determinable parameter, the local concentration of a second segment after the first of a pair is attached to the lattice. From combined data for these two association reactions a value of 10⁻⁴ M is obtained for this quantity. The large degree of segmental flexibility reported for the free heavy meromyosin is still manifested in the association with actin.     

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”.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

Thermal transitions of myosin and its helical fragments. Regions of structural instability in the myosin molecule.

The structural stabilities of all the familiar proteolytic fragments of myosin have been investigated in melting studies over the pH ranges 5.5-7.0 in 0.5 M KCl. All fragments except subfragment 2 undergo a melting transition manifested by the cooperative uptake of protons in the temperature range 34-47 degrees C, and these fragments experience an increase in transition temperature, Tm as the pH is increased. Subfragment 2 undergoes a melting transition in the 43-55 degrees C range, manifested by the dissociation of protons, and it experiences a decrease in Tm as the pH is increased. These results suggest that pH changes can modulate the relative stabilities of the light meromysin, subfragment-1, and subfragment-2 regions of the myosin molecule.