Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.     

N-ethylmaleimide-modified heavy meromyosin. A probe for actomyosin interactions

Treatment of rabbit skeletal muscle heavy meromyosin (HMM) with the sulfhydryl reagent N-ethylmaleimide (NEM) produces a species of HMM which remains tightly bound to actin in the presence of MgATP. NEM-HMM forms characteristic "arrowhead" complexes with actin which persist despite rinses with MgATP. NEM-HMM inhibits the actin activation of native HMM-ATPase activity, the superprecipitation of actomyosin, the contraction of glycerinated muscle myofibrils, and the contraction of cytoplasmic strands of the soil amoeba Chaos carolinensis. However, NEM-HMM does not interfere with in vitro microtubule polymerization or beating of demembranated cilia.     

Organization of native and in vitro-reassembled myosin filaments from lobster tonic muscle

Tonic muscle of the crusher claw of the American lobster (Homarus amencanus) was investigated with respect to sarcomeric organization and the capacity for self-assembly of extracted myosin for comparison with the same properties of rabbit muscle. Native myosin filaments in the lobster muscle are much longer than in rabbit skeletal fibers, and differ further in sarcomeric organization in showing an actinto-myosin relationship in which two actin filaments are shared between adjacent myosins in a 12-membered orbital. The self-assembly of lobster myosin into filaments comparable in length and fine structure to the natural filament was achieved in the presence of excess Mg²⁺, a condition not required for rabbit myosin self-assembly. Results of in situ and self-assembly studies indicate a difference in molecular organization between lobster and rabbit myosin filaments and of the inferred presence of regulatory factors in the formation of these ultrastructural elements. These studies represent the groundwork for an investigation of in vitro polymerization of actin in association with the synthetic lobster myosin filament.     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

Association of actin with the nuclear matrix from bovine lymphocytes

Nuclear matrix prepared from bovine lymphocytes contained a significant amount of actin. Both nuclear matrix actin and rabbit muscle actin showed the same electrophoretic mobility on SDS-gel. The matrix-associated actin could be separated into three isoproteins which may correspond to alpha-, beta- and gamma-actin. The most acidic spot of these isoproteins co-migrated with rabbit muscle actin (alpha-actin) on two-dimensional electrophoresis. The amino-acid composition of the nuclear matrix actin was closely related to that of rabbit muscle and to that of porcine brain actin. Moreover, the actin filaments, treated with 0.75 M guanidine hydrochloride, changed from the polymerized form of the nuclear matrix actin into a monomeric form (G-actin), which had strong inhibitor activity against pancreatic DNase I. From this inhibition, the actin content of the nuclear matrix was estimated to be about 12% of total matrix protein. When the nuclear matrix was digested with trypsin, the bulk of matrix protein was hydrolyzed, but about 80% of the actin remained associated with sphere structures (trypsin-treated nuclear matrix) precipitable by low speed centrifugation. SDS-gel analysis revealed that actin was one of the major components of the trypsin-treated nuclear matrix, which had a similar size and structure as the untreated nuclear matrix. The fibrogranular structure and residual nucleoli of the original nuclear matrix were well preserved against trypsin digestion; however, the peripheral lamina was removed. These results indicate that the matrix-associated actin is localized predominantly in the matrix interior, where it presumably interacts closely with the fibrogranular structure and/or the residual nucleoli.     

Effect of alkaloid sanguinarine and a pharmaceutical preparation ukrain on modulation of vesicular membrane fusion and actin cytoskeleton of macrophages

A study was made of modulations of lysosome-phagosome fusion process and of fibrillar actin content in mouse peritoneal macrophages by an antitumor alkaloid sanguinarine and a derivative drug Ukrain. In addition, effects of these substances on in vitro polymerization of monomeric globular actin from rabbit muscle were investigated. Sanguinarine and Ukrain stimulated lysosome-phagosome fusion and increased the content of polymerized fibrillar form of actin in mouse macrophages. Effects of these substances were enhanced at their higher concentrations. Both sanguinarine and Ukrain induced in vitro polymerization of globular actin from rabbit muscle. A possible role of sanguinarine and Ukrain in changing vesicular membrane states during intracellular membrane interaction in lysosome-phagosome fusion process was discussed. The influence of these substances on actin polymerization and actin cytoskeleton rearrangement was evaluated. It could be supposed that sanguinarine and Ukrain may alter intracellular membrane transport.     

The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation.

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins--bovine aorta actin and chicken gizzard actin--differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably cloer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

Tension development in skinned glycerinated rabbit psoas fiber segments irrigated with soluble myosin fragments

Single glycerinated rabbit psoas muscle fibers were skinned by splitting them lengthwise. The fiber segments thus obtained were more easily accessible to solutes in the surrounding medium than the intact fibers. Using such segments, active tension could be fully abolished by adding N-ethylmaleimide under conditions which lead to inhibition of actin activation of the ATPase activity of myosin. Such muscles could, however, develop tension after irrigation with myosin or with the water-soluble active myosin fragments heavy meromyosin (HMM) or its subfragment 1 (HMM-S1). The induced tensions increased with increasing protein concentration in the irrigating solution. At any given protein concentration, the tension generated by myosin was larger than that produced by HMM which was, in turn, greater than that induced by HMM-S1 e.g. at 15 mg/ml protein the tensions produced by these three myosin moieties were 44.0, 14.0 and 2.8 g/cm², respectively. The tension was found to be intimately associated with ATP splitting; thus, HMM and HMM-S1 which have been treated with reagents abolishing actin-activated ATPase failed to induce tension development. A contractile force may thus be generated through the interaction with actin of the water-soluble, enzymatically active, myosin subfragments involving the splitting of ATP.     

Sarcomere structures in the rabbit psoas muscle as revealed by fluorescent analogs of phalloidin

Summary The fluorescent analogs of phalloidin (rhodamine-and fluorescein-phalloidin) bind tightly to the skinned fibres of rabbit psoas muscle at essentially the same sites as phalloidin and mainly stain the known regions of actin localization in the sarcomere: the thin filaments and Z bands. On both sides of the Z bands, unstained zones were observed, suggesting the presence of proteins tightly bound to the thin filaments. In myofibrils which are stretched to such an extent that the actin and myosin filaments do not overlap, stained bands could also be seen at the myosin-band border, which suggests the localization of actin at these sites.     

Comparative Study on Interaction of DNaseⅠ with Pollen Actin and Rabbit Muscle Actin

Actin purified from maize pollen grains like actin from other sources could considerably inhibit the activity of DNase Ⅰ . A linear relationship existed between inhibition and the concentration of actin. However, DNase Ⅰ was less inhibited by pollen actin than by rabbit muscle actin under the same conditions. The values of Kapp of inhibition were 1.25 μg/mL for pollen actin and 0.75 μg/mL for rabbit muscle actin. DNase Ⅰdepolymerized both pollen and rabbit muscle actin filaments. But the rate of depolymerization of pollen F-actin was higher than that of rabbit muscle F-actin under the same conditions.     

玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的比较研究

本文对玉米花粉肌动蛋白和兔骨骼肌肌动蛋白进行了比较研究。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白具有相同的分子量(42KD)。玉米花粉肌动蛋白可与兔抗鸡胃肌动蛋白抗血清产生免疫沉淀反应。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的氨基酸组成以及胰蛋白酶水解所得到的肽谱都相似。它们的羧基未端氨基酸顺序完全一致,其顺序都是Lys.Cys.Phe(COOH)。它们的圆二色谱基本相同,由圆二色谱计算得到的二级结构数据也相近。以上的结果表明了玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的相似性。     

Activation of the Adenosine Triphosphatase of Limulus polyphemus Actomyosin by Tropomyosin

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.     

Actin in the green algae Coleochaete and Mougeotia

Summary Filaments associated with chloroplasts in cytoplasmic homogenates of the algae Coleochaete scutata and Mougeotia sp. bind rabbit skeletal muscle heavy meromyosin (HMM) to form arrowhead complexes that could be dissociated with ATP. This result suggests that the filaments are actin which may be involved in the characteristic chloroplast movements exhibited by these algae.     

Comparison of the effects of smooth and skeletal muscle actins on smooth muscle actomyosin Mg2+-ATPase

Actin has been purified from smooth muscle (chicken gizzard) by two different procedures and its activation of smooth muscle myosin Mg2+-ATPase activity compared with that achieved with rabbit skeletal muscle actin. The procedure of Pardee and Spudich (Methods Enzymol. (1982) 85, 164-181) for the purification of rabbit skeletal muscle actin is readily applicable to the isolation of chicken gizzard actin, enabling large quantities to be purified in two days. Smooth muscle actin could be successfully stored as F-actin at -80 degrees C and survived freezing and thawing at least twice. Smooth muscle actin activated myosin Mg2+-ATPase to a higher level than its skeletal muscle counterpart (77.9 nmol Pi/min/mg myosin vs 48.1 nmol Pi/min/mg myosin).     

Direct measurement of VDAC-actin interaction by surface plasmon resonance

VDAC - a mitochondrial channel involved in the control of aerobic metabolism and apoptosis - interacts in vitro and in vivo with a wide repertoire of proteins including cytoskeletal elements. A functional interaction between actin and Neurospora crassa VDAC was reported, excluding other VDAC isoforms. From a recent genome-wide screen of the VDAC interactome, we found that human actin is a putative ligand of yeast VDAC. Since such interaction may have broader implications for various mitochondrial processes, we probed it with Surface Plasmon Resonance (SPR) technology using purified yeast VDAC (YVDAC) and rabbit muscle G-actin (RGA). We show that RGA binds to immobilized YVDAC in a reversible and dose-dependent manner with saturating kinetics and an apparent K_D of 50 μg/ml (1.2 μM actin). BSA does not bind VDAC regardless of the concentrations. Alternatively, VDAC binds similarly to immobilized RGA but without saturating kinetics. VDAC being known to interact with itself, this latter interaction was directly measured to interpret the RGA signals. VDAC could bind to VDAC without saturating kinetics as expected if higher order binding occurred, and could account for maximally 66% of the non-saturating behavior of VDAC binding onto RGA. Hence, actin-VDAC interactions are not a species-specific oddity and may be a more general phenomenon, the role of which ought to be further investigated.     

Peptide mapping of contractile proteins: Two-dimensional analysis of cyanogen bromide fragments on polyacrylamide gels

Peptide mapping of contractile proteins is necessary for correlation of the structual properties of these molecules with their distinct roles in various cell types. The large size of several of these polypeptides requires their cleavage by cyanogen bromide and the separation of the resulting products on a two-dimensional polyacrylamide-gel system to ensure reasonably complete peptide maps. Such a peptide map of rabbit skeletal muscle actin is in agreement with predictions from the amino acid sequence. The peptide map of rabbit skeletal muscle myosin can be resolved into maps of heavy and light meromyosins resulting from limited tryptic digestion of the myosin. Unique regions of the myosin map are occupied by one or the other of these segments. Analysis of native 105,000 M_r elam adductor paramyosin and its 94,000 M_r proteolytic products indicates that specific changes in peptide composition have occurred.     

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

Myosin content and filament structure in smooth and striated muscle

Fibres from four different muscles (rabbit psoas, guinea pig taenia coli, Lethocerus flight and leg) were glycerol-extracted, homogenized and dissolved in a sodium dodecyl sulphate solution. The relative mass of the myosin heavy chain and actin polypeptides present in these extracts was measured by polyacrylamide gel electrophoresis. The ratio was found to be consistent for each muscle and to differ widely between muscles. The results were used to calculate the number of myosin molecules per subunit repeat along the thick filaments of the striated muscles and ribbon-like filaments, and so to test a theory of filament structure.     

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

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.