Porcine platelet tropomyosin: Purification,characterization and paracrystal formation

Porcine platelet tropomyosin has been isolated by hydroxyapatite chromatography following isoelectric precipitation and ethanol fractionation. A single component (M_r = 30,000 on polyacrylamide gel electrophoresis in sodium dodecyl sulphate) was obtained in a variety of gel electrophoretic systems, including urea/sodium dodecyl sulphate and isoelectric focussing, suggesting the presence of a single polypeptide species. This contrasts with the observations by others using horse platelet tropomyosin or tropomyosins isolated from brain, where two polypeptides were found. The amino acid composition of porcine platelet tropomyosin was virtually identical to that of the horse platelet protein except for the presence of a single cysteine residue in the pig protein, whereas horse tropomyosin contains two. Oxidation of this sulphydryl group produced a molecule of over 60,000 M_r on polyacrylamide gels in sodium dodecyl sulphate, suggesting by analogy with skeletal muscle tropomyosin that the two chains had been linked and therefore existed in register in the coiled-coil structure. Cleavage at the cysteine produced a fragment of M_r = 19,000, indicating that this residue was located about one-third of the distance from a molecular end.Magnesium paracrystals of platelet tropomyosin were examined by electron microscopy following negative staining and found to have a repeat of 345 Å, close to that expected for an extended alpha-helical coiled-coil with an apparent M_r of 30,000. The repeating unit was centrosymmetric and the appearance of broken paracrystals suggested that the molecular ends lie on a dyad axis. Location of the sulphydryl residues in the paracrystals by labelling with N-pyrrolo-isomaleimide showed two bands separated by approximately 80 Å, which was consistent with the location of the molecular ends on a dyad.The amount of tropomyosin present was estimated as 2·2% of the total platelet protein. This implied a molar ratio of G-actin to tropomyosin of about 14:1, based on previous estimates of the actin content in porcine platelets. Assuming that one molecule of tropomyosin will bind six molecules of actin (based on the reduced molecular length of the platelet protein), there was not sufficient tropomyosin to bind to more than half the total actin in the cell.     

Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

Kinetics of steady state ATPase activity and rigor complex formation of acto-heavy meromyosin

1. Actin and heavy meromyosin, initially mixed in a Mg-ATP solution, began to form the rigor complex slowly after ATP in the solution had been completely hydrolyzed.

2. This was because the heavy meromyosin-product complex formed via ATP hydrolysis was almost completely dissociated from actin even in the absence of ATP and as soon as this heavy meromyosin-product complex was decomposed, the heavy meromyosin combined with actin forming the rigor complex.

3. Linear plots were obtained when the reciprocal of the excess rate of the actin-accelerated rigor complex formation was plotted against the reciprocal of the added actin concentration as similar with those made on the steady acto-heavy meromyosin ATPase.

4. The V of the rigor complex formation process was about 1/5 of that of the steady acto-heavy meromyosin ATPase activity, showing that the actomyosin ATPase activity could not be explained merely by the actin-accelerated decomposition of the heavy meromyosin-product complex.

5. The same analyses were carried out on myosin subfragment 1.

6. Our results could be explained by considering the two non-identical active sites of myosin, and we propose the following scheme for the actomyosin ATPase.

7. Actin accelerates the rate-limiting bond hydrolysis in the ATPase occurring at one active site of myosin, as well as the rate-limiting decomposition of the heavy meromyosin-product complex formed at another site.     

Actin paracrystal in the acrosome of newt sperm

When a newt sperm-head was treated with trypsin and DNase, an arrow-like thin rod was revealed. This rod presumably corresponds to the ‘perforatorium’ described by Picheral [1, 2] in Pleurodele sperm. It consisted of one apical and one caudal part. In the apical part there appeared to be an envelope with a 530 Å structural repeat, inside which coursed a filament bundle, presumably identical with that in the caudal part. In the caudal part, a characteristic filament bundle, quite similar to the paracrystal of rabbit skeletal actin [3], was observed after extensive treatment with trypsin. The optical diffraction pattern of this bundle indicates that it has the same helical symmetry as that of rabbit skeletal actin [4] but slightly different from that of the acrosomal process of Limulus sperm [5]. The diffraction pattern frequently has a strong meridional reflection at about (27 Å)⁻¹, which is usually observed only with low intensity in the actin paracrystals. This fact suggests that the structural unit in the bundle has a shape considerably different from that of the usual G-actin.     

Subunit size and paracrystal structure of avian liver acetyl-CoA carboxylase.

The subunit molecular weight of chicken liver acetyl-CoA carboxylase has been redetermined by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. In the presence of parotid trypsin inhibitor, the immunoprecipitate gave a single band corresponding to a molecular weight of 230,000, which was also found to contain bound biotin. From the biotin content of the protomer (1.0 prosthetic group per 480,000 daltons) it appears that it consists of two non identical subunits, both with molecular weights of approximately 230,000.Electron microscopy has been carried out on the active filamentous form of the enzyme and on paracrystals formed under high-salt conditions. These indicate that the filaments are readily distortable helical ribbons, with an approximate axial repeat of 1100 Å, containing eight protomers. The paracrystals are made up of a staggered lateral packing of filaments.     

Actin paracrystal induction by forskolin and by db-cAMP in CHO cells

Forskolin, a hypotensive diterpine, is assumed to be a potent activator of adenylate cyclase leading to increased levels of cAMP. When this drug is used at 10(-5) M on CHO-C14 cells in culture, it induces within 15 min actin paracrystals in all cells. At this time the paracrystals are mostly situated close to the cell periphery. Electron microscopy (EM) shows structures typical of actin paracrystals. Scanning electron microscopy (SEM) reveals a reduction in surface microvilli and blebs. Identical results can be obtained by adding 1 mM db-cAMP to the culture medium directly. The paracrystals are observed within 15 min and thus represent one of the earliest ultrastructural changes so far described for reverse transformation of CHO cells by db-cAMP. The microtubular and vimentin profiles appear unchanged by forskolin treatment of CHO-K1 cells. Out of currently unknown reasons forskolin does not induce the actin transformation in several other commonly used cell lines.     

Modeling the lateral organization of collagen molecules in fibrils using the paracrystal concept

A characteristic feature of the dense phases formed by fiber-shaped molecules is their organization into parallel rods packed in a hexagonal or pseudo-hexagonal lateral network. This is typically the case for the collagen triple helices inside fibrils, as confirmed by recent X-ray diffraction experiments carried out on highly crystallized fibers obtained by immersing the freshly extracted fibers in a salt-controlled medium. However such diffraction patterns also generally exhibit additional features in the form of diffuse scattering, which is a clear signature of a low degree of lateral ordering. Only few studies have analyzed and modeled the lateral packing of collagen triple helices when the structure is disordered. Some authors have used the concept of short-range order but this approach does not contain any echo of a hexagonal order. In this study, we use an analytical expression derived from the paracrystal model which retains the hexagonal symmetry information and leads to a good agreement with the experimental data in the medium-angle region. This method is quite sensitive to the degree of disorder and to the inter-object distance. One clear result is that the shift in peak positions, generally attributed to variations in intermolecular distances, can also arise from a change in the degree of ordering without any significant modification of the distances. This underlines the importance of evaluating the degree of ordering before attributing a shift in peak position to a change in the unit-cell. This method is generic and can be applied to any system composed of rod-shaped molecules.