scholarly journals Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ.

1975 ◽  
Vol 66 (3) ◽  
pp. 508-520 ◽  
Author(s):  
L G Tilney ◽  
P Detmers
Keyword(s):  
Molecular Weight ◽  
Actin Filaments ◽  
Cross Linking ◽  
Muscle Actin ◽  
Lateral Movement ◽  
Erythrocyte Ghosts ◽  
Heavy Meromyosin ◽  
Net Charge ◽  
Muscle Myosin

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.

scholarly journals THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

1973 ◽  
Vol 59 (1) ◽  
pp. 109-126 ◽  
Author(s):  
Lewis G. Tilney ◽  
Sadashi Hatano ◽  
Harunori Ishikawa ◽  
Mark S. Mooseker
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acetone Powder ◽  
Heavy Meromyosin ◽  
Skeletal Muscle Myosin ◽  
Sperm Extract ◽  
Triton X ◽  
Muscle Myosin

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.

scholarly journals Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.

1975 ◽  
Vol 64 (2) ◽  
pp. 289-310 ◽  
Author(s):  
L G Tilney
Keyword(s):  
Molecular Weight ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Stable States ◽  
Equimolar Ratio ◽  
Net Charge ◽  
Molecular Weights ◽  
Acrosomal Reaction ◽  
Sperm Motion ◽  
Filament Bundle

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.

scholarly journals Actin filament destruction by osmium tetroxide

1978 ◽  
Vol 77 (3) ◽  
pp. 837-852 ◽  
Author(s):  
P Maupin-Szamier ◽  
TD Pollard
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Sodium Phosphate ◽  
Osmium Tetroxide ◽  
Time Span ◽  
Cross Linking ◽  
Muscle Actin ◽  
Viscosity Change ◽  
Sodium Phosphate Buffer ◽  
Sulfur Containing

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

scholarly journals FORMATION OF ARROWHEAD COMPLEXES WITH HEAVY MEROMYOSIN IN A VARIETY OF CELL TYPES

1969 ◽  
Vol 43 (2) ◽  
pp. 312-328 ◽  
Author(s):  
Harunori Ishikawa ◽  
Richard Bischoff ◽  
Howard Holtzer
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Cell Types ◽  
Nerve Cells ◽  
Collagen Fibrils ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Cellular Components

Heavy meromyosin (HMM) forms characteristic arrowhead complexes with actin filaments in situ. These complexes are readily visualized in sectioned muscle. Following HMM treatment similar complexes appear in sectioned fibroblasts, chondrogenic cells, nerve cells, and several types of epithelial cells. Thin filaments freshly isolated from chondrogenic cells also bind HMM and form arrowhead structures in negatively stained preparations. HMM-filament complexes are prominent in the cortex of a variety of normal metaphase and Colcemid-arrested metaphase cells. There is no detectable binding of HMM with other cellular components such as microtubules, 100-A filaments, tonofilaments, membranes, nuclei, or collagen fibrils. The significance of HMM-filament binding is discussed in view of the finding that arrowhead complexes form in types of cells not usually thought to contain actin filaments.

A comparison of isolated and in situ thick filaments from smooth muscle

1986 ◽  
Vol 44 ◽  
pp. 156-157
Author(s):  
E.L. Buhle ◽  
A.V. Somlyo ◽  
A.P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Muscle Actin ◽  
Thin Sections ◽  
Ultrastructural Studies ◽  
Thick Filaments ◽  
Myosin Filaments ◽  
Rabbit Portal Vein ◽  
Stable Structures

Early ultrastructural studies of smooth muscle are consistent with the sliding filament mechanism of contraction. Myosin filaments are stable structures in situ and can be found in both relaxed and contracted muscle. Actin filaments can be decorated with SI subfragments of myosin to show a polarity similar to the Z-lines of skeletal muscle. The work presented here is a comparison of isolated thick filaments from relaxed chick amnion with thick filaments obtained in situ from longitudinal thin sections (∽50nm thick) of rabbit portal vein in rigor.

Actin from Saccharomyces cerevisiae

1982 ◽  
Vol 2 (10) ◽  
pp. 1270-1278
Author(s):  
C Greer ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Filamentous Actin ◽  
Heavy Meromyosin ◽  
Proteolytic Fragment ◽  
Maximal Activation ◽  
Final Fraction ◽  
Muscle Myosin

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.

scholarly journals A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments.

1984 ◽  
Vol 99 (1) ◽  
pp. 71-78 ◽  
Author(s):  
C M Goodloe-Holland ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Dictyostelium Discoideum ◽  
Electron Micrographs ◽  
Actin Filaments ◽  
Lateral Movement ◽  
Heavy Meromyosin ◽  
Membrane Cytoskeleton ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The binding between sonicated Dictyostelium discoideum plasma membrane fragments and F-actin on Sephacryl S-1000 beads was found to be competitively inhibited by myosin subfragment-1. This inhibition is MgATP-sensitive, exhibits a Ki of approximately 5 X 10(-8) M, and is reciprocal, since membranes inhibit the binding of 125I-heavy meromyosin to F-actin on beads. These experiments demonstrate that membrane binding and S-1 binding to F-actin on beads are mutually exclusive and, therefore, that the membrane fragments bind predominantly to the sides, rather than to the ends, of the actin filaments. This conclusion is supported by electron micrographs that show many lateral associations between membrane fragments and bead-associated actin filaments. Such lateral associations could play an important role in the organization and lateral movement of membrane proteins by the cytomusculature.

scholarly journals 3-Methylhistidine in actin and other muscle proteins

1967 ◽  
Vol 105 (1) ◽  
pp. 361-370 ◽  
Author(s):  
P. Johnson ◽  
C I Harris ◽  
S V Perry
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Adult Rabbit ◽  
Muscle Actin ◽  
Muscle Proteins ◽  
Heavy Meromyosin ◽  
Tryptic Digest ◽  
Peptide Fraction

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7·6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.

Cross-linking of actin filaments by heavy meromyosin

1979 ◽  
Vol 133 (4) ◽  
pp. 549-556 ◽  
Author(s):  
John Trinick ◽  
Gerald Offer
Keyword(s):  
Actin Filaments ◽  
Cross Linking ◽  
Heavy Meromyosin
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