Functional Characterisation of Dictyostelium Myosin II with Conserved Tryptophanyl Residue 501 Mutated to Tyrosine

AbstractWe created aDictyostelium discoideummyosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in aDictyosteliumstrain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin inDictyostelium. Additionally, we expressed the W501Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.

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The distribution of myosin II in Dictyostelium discoideum slug cells.

The Journal of Cell Biology ◽

10.1083/jcb.115.5.1267 ◽

1991 ◽

Vol 115 (5) ◽

pp. 1267-1274 ◽

Cited By ~ 12

Author(s):

S Eliott ◽

P H Vardy ◽

K L Williams

Keyword(s):

Cell Motility ◽

Dictyostelium Discoideum ◽

Immunofluorescence Microscopy ◽

Molecular Genetic ◽

Myosin Ii ◽

Three Dimensional ◽

Homogeneous Distribution ◽

Multicellular Development ◽

Insight Into

While the role of myosin II in muscle contraction has been well characterized, less is known about the role of myosin II in non-muscle cells. Recent molecular genetic experiments on Dictyostelium discoideum show that myosin II is necessary for cytokinesis and multicellular development. Here we use immunofluorescence microscopy with monoclonal and polyclonal antimyosin antibodies to visualize myosin II in cells of the multicellular D. discoideum slug. A subpopulation of peripheral and anterior cells label brightly with antimyosin II antibodies, and many of these cells display a polarized intracellular distribution of myosin II. Other cells in the slug label less brightly and their cytoplasm displays a more homogeneous distribution of myosin II. These results provide insight into cell motility within a three-dimensional tissue and they are discussed in relation to the possible roles of myosin II in multicellular development.

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Synthesis and solution conformation of [Trp1, Val5]-angiotensin II

Canadian Journal of Biochemistry ◽

10.1139/o77-013 ◽

1977 ◽

Vol 55 (1) ◽

pp. 75-82 ◽

Cited By ~ 16

Author(s):

Peter W. Schiller

Keyword(s):

Angiotensin Ii ◽

Solid Phase ◽

Fluorescence Emission ◽

Resonance Energy ◽

Tryptophan Fluorescence ◽

Exchange Chromatography ◽

Phase Method ◽

Aqueous Environment ◽

Solution Conformation ◽

Intramolecular Distance

[Trp1, Val5]-Angiotensin II was synthesized by the solid-phase method and purified by partition chromatography on Sephadex G-25 and by ion-exchange chromatography on Sephadex SP-25. Relative to [Val5]-angiotensin II, the analog displayed 36% smooth muscle activity in a preparation of the superior mesenteric artery. Conformational aspects of the analog were revealed by fluorescence techniques. The fluorescence emission maximum at 350 nm suggests a completely aqueous environment for the tryptophanyl residue in [Trp1, Val5]-angiotensin II. Singlet–singlet resonance energy transfer between Tyr in position 4 and Trp in position 1 was evaluated for a calculation of the intramolecular distance between these two residues on the basis of the Förster equation. From the relative increase of tryptophan fluorescence a transfer efficiency of > 0.9 was obtained, which is compatible with the observed complete quenching of tyrosine fluorescence in the analog. The computed average intramolecular distance of < 8 Å precludes an extended conformation for the N-terminal sequence encompassing residues 1 through 4 at neutral pH and suggests the existence of a loop in this part of the molecule. The results are discussed in relation to the various models proposed for the solution conformation of angiotensin II.

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Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium

Journal of Cell Science ◽

10.1242/jcs.112.13.2195 ◽

1999 ◽

Vol 112 (13) ◽

pp. 2195-2201 ◽

Cited By ~ 1

Author(s):

S. Shu ◽

R.J. Lee ◽

J.M. LeBlanc-Straceski ◽

T.Q. Uyeda

Keyword(s):

Myosin Head ◽

Myosin Ii ◽

Equatorial Region ◽

Chain Gene ◽

Cleavage Furrow ◽

Tail Domain ◽

Specific Domain ◽

Dictyostelium Myosin ◽

Skeletal Myosin

Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for Mydelta943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.

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Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis

Journal of Cell Science ◽

10.1242/jcs.108.3.1105 ◽

1995 ◽

Vol 108 (3) ◽

pp. 1105-1115 ◽

Cited By ~ 9

Author(s):

E. Shelden ◽

D.A. Knecht

Keyword(s):

Cell Morphology ◽

Mutant Cell ◽

Myosin Ii ◽

Cell Phenotype ◽

Computer Assisted ◽

Wild Type ◽

Isolated Cells ◽

Multicellular Development ◽

Periodic Movement ◽

Mutant Cells

We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 microns/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 microns/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5 +/- 2.0 microns/min, while wild-type cells under these conditions move at 5.9 +/- 4.5 microns/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.

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Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

Biochemical Journal ◽

10.1042/bj1230757 ◽

1971 ◽

Vol 123 (5) ◽

pp. 757-771 ◽

Cited By ~ 48

Author(s):

R. R. Eady ◽

P. J. Large

Keyword(s):

Fluorescence Emission ◽

Electrophoretic Analysis ◽

Enzyme Mechanism ◽

Heat Denaturation ◽

Kinetic Properties ◽

Microbial Oxidation ◽

Prosthetic Group ◽

Iron And Zinc ◽

Amine Dehydrogenase ◽

Phenazine Methosulphate

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver–Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.

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Different modes of interaction of pulmonary surfactant protein SP-B in phosphatidylcholine bilayers

Biochemical Journal ◽

10.1042/bj3270133 ◽

1997 ◽

Vol 327 (1) ◽

pp. 133-138 ◽

Cited By ~ 33

Author(s):

Antonio CRUZ ◽

Cristina CASALS ◽

Kevin M. W. KEOUGH ◽

Jesús PÉREZ-GIL

Keyword(s):

Aqueous Phase ◽

Protein Interactions ◽

Pulmonary Surfactant ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Egg Yolk ◽

Surfactant Protein ◽

Scanning Calorimetry ◽

Adsorption Activity ◽

Egg Yolk Phosphatidylcholine

Pulmonary surfactant-associated protein B (SP-B) has been incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) by two different procedures to characterize the dependence of lipid–protein interactions on the method of reconstitution. In method A the protein was dissolved in a small volume of either methanol or 60% (v/v) acetonitrile and injected into an aqueous phase containing phospholipid vesicles. In method B the vesicles were prepared by injection of a mixture of phospholipid and SP-B dissolved in methanol or aqueous acetonitrile. Both methods of reconstitution led to the extensive interaction of SP-B with PC bilayers as demonstrated by co-migration during centrifugation, marked protection against proteolysis, change in the fluorescence emission intensity of SP-B, and protection of SP-B tryptophan fluorescence from quenching by acrylamide. SP-B promoted the rapid adsorption of DPPC on an air/liquid interface irrespective of the method of protein reconstitution. However, the interfacial adsorption activity of SP-B reconstituted by method B remained stable for hours, but that of SP-B prepared by method A decreased with time. Electron microscopy showed that the injection of SP-B into an aqueous phase containing PC or DPPC vesicles (method A) induced a rapid aggregation of vesicles. By contrast, a much longer time was required for detecting vesicle aggregation when the protein was reconstituted by co-injection of SP-B and phospholipids (method B). The presence of 5% (w/w) SP-B in DPPC bilayers prepared by method B broadened the differential scanning calorimetry thermogram and decreased the enthalpy of the transition. In contrast, the injection of SP-B into preformed DPPC vesicles (method A) did not influence the gel-to-liquid phase transition of DPPC bilayers. Taken together, these results indicate that the mode and extent of interaction of SP-B with surfactant phospholipids depends on the conditions of preparation of lipid/protein samples, and that care should be taken in the interpretation of findings from reconstituted systems on the role of these surfactant proteins in the alveolar space.

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In Vitro Study of Mechanical and Kinetic Properties of Myosin II from Frog Skeletal Muscle

Biophysical Journal ◽

10.1016/j.bpj.2008.12.3248 ◽

2009 ◽

Vol 96 (3) ◽

pp. 614a

Author(s):

Ravikrishnan Elangovan ◽

Marco Capitanio ◽

Francesco S. Pavone ◽

Vincenzo Lombardi

Keyword(s):

Skeletal Muscle ◽

Myosin Ii ◽

In Vitro Study ◽

Vitro Study ◽

Kinetic Properties ◽

Frog Skeletal Muscle

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The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

Biochemical Journal ◽

10.1042/bj2430047 ◽

1987 ◽

Vol 243 (1) ◽

pp. 47-54 ◽

Cited By ~ 22

Author(s):

L J Larsson ◽

P Lindahl ◽

C Hallén-Sandgren ◽

I Björk

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Cross Linking ◽

Bait Region ◽

Close Proximity ◽

Tryptophan Residues ◽

Thioester Bond ◽

Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

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Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

Biochemistry and Cell Biology ◽

10.1139/o04-131 ◽

2005 ◽

Vol 83 (2) ◽

pp. 109-114 ◽

Cited By ~ 5

Author(s):

Hong-Min Tang ◽

Hong Yu

Keyword(s):

Fluorescence Intensity ◽

Tertiary Structure ◽

Structural Characteristics ◽

Molten Globule ◽

Guanidine Hydrochloride ◽

Arginine Kinase ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Blue Shift ◽

Molten Globule State

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

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