Isoforms of myosin and actin in human, monkey and rat myometrium. Comparison of pregnant and non-pregnant uterus proteins

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.     

Two-dimensional gel electrophoresis of chicken skeletal muscle proteins with agarose gels in the first dimension

An improved method of two-dimensional gel electrophoresis is described. The method is specifically developed for preparing a “protein map” of chicken skeletal muscle, and is found to be applicable to the analysis of most protein constituents including high molecular ones, such as myosin heavy chain, without using any detergents in the first dimension. Omission of detergents from the focusing medium results in two advantages. (i) The first-dimension isoelectric focusing pattern can be recorded by taking a photograph of the gel prior to the second-dimension electrophoresis, so that even a close doublet band in the first dimension, which forms one spot in the second dimension, can be found heterogeneous in component by examining the first-dimension pattern of the same gel. (ii) Since peptides of relatively large molecular weights can be analyzed by first-dimension isoelectric focusing, complex formation between polypeptides with different isoelectric points is demonstrable. For example, troponin T, troponin I, and troponin C are found by two-dimensional gel electrophoresis to form a complex in a 4 m urea solution, and so are troponin I and troponin C in a 5 m urea solution.     

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Purification of uterine myosin and synthetic filament formation

Rabbit uterine myosin was purified by DEAE-Sephadex column chromatography. The purified myosin was shown to be free from contamination with actin or other impurities by sodium dodecyl sulfate gel electrophoresis. It was also shown to have two light chains with molecular weights of 22,000 and 17,000. The C protein normally found in crude, rabbit skeletal muscle myosin was not found in crude, rabbit uterine myosin.On reducing the ionic strength of a solution of rabbit uterine myosin, the myosin molecules first aggregated to form short, tapered bipolar filaments with bare zones. These were observed in the presence or absence of 10 mm-magnesium. These filaments ranged in length from 0.3 μm to 0.6 μm. On reaching 0.1 m-KCl, and only if 10 mm-magnesium was present, the filaments grew in length by linear overlap of the tapered bipolar filaments and obliteration of the bare zone regions. These filaments ranged in length from 0.7 μm to 1.2 μm.     

Identification of Actin in Highly Purified Synaptic Vesicles from the Electric Organ of Torpedo marmorata

Abstract: Evidence has been obtained that actin is a major constituent of highly purified synaptic vesicles isolated from the electric organ of Torpedo marmorata . The mobility of a prominent spot in the polypeptide pattern of vesicles in high-resolution two-dimensional polyacrylamide gel electrophoresis is very similar to the mobility of the main component in the actin preparation purified from the whole electric organ by affinity chromatography on immobilized pancreatic deoxyribonuclease I. The comparison of tryptic peptide maps obtained from the putative vesicle actin and authentic actin from the electric organ, both purified by two-dimensional gel electrophoresis and labeled in situ with ¹²⁵I, showed about 88% homology, thereby supporting the conclusion that the vesicle actin is indeed an actin isoform.     

Cytoskeletal functions of cytoplasmic contractile proteins

This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system inthe cytoplasmic matrix. Biochemical experiments show that cycoplasmic actin filaments can form a solid gel under conditions likely to exist in living cells. The actin filaments are associated with other proteins which may stabilize the gel and which are involved with motile force generation like myosin. Ultrastructural studies show that actin filaments are difficult to preserve, but that under stabilizing conditions networks of actin filaments are found throughout the cytoplasmic matrix.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

The cortical actin cytoskeleton of unactivated zebrafish eggs: Spatial organization and distribution of filamentous actin,nonfilamentous actin,and myosin-II

Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization. © 1996 Wiley-Liss, Inc.     

An isoelectric focusing study of plasma membrane actin.

Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [³⁵S]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The α, β, and γ isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of ³⁵S into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.     

The two actin-binding regions on the myosin heads of cardiac muscle

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.     

Characterization and purification of lupus antigen La, and RNA-binding protein.

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.     

Myosin and actin in melanophore-like variants of goldfish erythrophoroma cells: their intracellular distribution and possible association with pigment translocation

Summary The occurrence and intracellular distribution of myosin and actin in melanophore-like cells derived from a goldfish erythrophoroma cell line have been studied by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunofluorescence using antisera against chick gizzard myosin heavy chain and carp skeletal muscle actin. SDS-PAGE of the cell extracts separates out one band at 200 kDalton; this is conjugated with the anti-myosin antiserum. Immunofluorescence using the anti-myosin antiserum discloses that myosin in these cells occurs in two forms: discrete, minute clusters and thin filaments bearing a resemblance to stress fibers. The former is distributed evenly over the entire cytoplasm in the cells with dispersed pigments and, upon pigment aggregation, accumulates densely around collapsed melanosomes. The latter runs as thin bundles either radially along the cell center-to-periphery axis or connecting the corners of cell margins; it gives a similar profile in all states of the motile response. Immunofluorescence using the antiactin antiserum or rhodamine-conjugated phalloidin discloses that actin is similarly distributed to myosin, suggesting its possible existence as actomyosin. Simultaneous translocation of the amorphous forms of myosin and actin with melanosomes indicates that they may be involved in pigment migration.     

Purification of native myosin filaments from muscle

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.     

Heterogeneity of renin substrate released from hepatocytes and in brain extracts

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17β estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000–65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.     

The identification of myosin in rabbit hepatocytes.

A myosin-like protein was identified in isolated rabbit liver cells. It was extracted with high-ionic-strength buffer containing ATP, and purified by gel filtration in the presence of iodide. The myosin polypeptide was indistinguishable in size from the heavy chain of muscle myosin as determined by electrophoresis on polyacrylamide gels and gel filtration in the presence of sodium dodecyl sulfate. The hepatic myosin had an amino acid composition similar to that of muscle myosin, but lacked 3-methylhistidine. The Mg2+ -ATPase of the myosin was not activated by muscle actin. At low ionic strength, in the presence of Mg2+, the protein aggregated to form bipolar filaments 0.3 mum in length. A protein which resembled muscle actin in size and amino acid composition was extracted along with the myosin. Based on scans of stained sodium dodecyl sulfate polyacrylamide gels, the myosin content was estimated as 0.3% to 0.4% of the cell protein. The actin-like component was present in approximately ten-fold excess by weight. This ratio suggests that the organization and function of myosin in the hepatocyte is very different from that in the muscle cell.