In postmeiotic male germ cells poly (A) shortening accompanies translation of mRNA encoding γ enteric actin but not cytoplasmic β and γ actin mRNAs

In the mammalian testis the cytoplasmic β and γ actins are expressed in all stages of germ-cell differentiation, whereas γ enteric actin is expressed in germ cells solely in postmeiotic stages. Northern blot analysis of mouse testicular RNAs reveals actin mRNAs of about 2.1, 1.5, and 1.4 kB. The 2.1-kB mRNAs encode the cytoplasmic β and γ actins, whereas the two faster-migrating actin mRNAs encode γ enteric actin. When post-mitochondrial mouse testis extracts are fractionated by sucrose gradient centrifugation, the 1.5-kB γ enteric actin mRNA is primarily found in the nonpolysomal fraction, whereas the 1.4-kB γ enteric actin is polysomal. When the poly (A) tails are removed, the nonpolysomal and polysomal γ enteric actin mRNAs both migrate at 1.3 kB, indicating that the difference in electrophoretic mobilities of the two γ enteric actin mRNAs is caused by poly (A) length differences. The nonpolysomal and polysomal forms of the cytoplasmic β and γ actins show similar electrophoretic mobilities before and after deadenylation. Sequence comparison of the 3′ untranslated region of the mouse γ enteric actin to the 3′ untranslated regions of other testicular mRNAs that undergo partial deadenylation reveals three highly-conserved sequence elements. These data demonstrate that the poly (A) shortening of polysomal mRNAs previously seen only with testis-specific mRNAs that are stored as mRNPs also occurs with mRNAs of widely-expressed genes that are expressed in postmeiotic male germ cells. The mRNAs all contain specific conserved sequence elements in their 3′ untranslated regions. © 1996 Wiley-Liss, Inc.     

Post-translational processing of the amino terminus affects actin function

We have studied the importance of N-terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two-dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin-layer electrophoresis we have shown that bestatin [( 3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine), an inhibitor of aminopeptidases, can inhibit actin N-terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non-polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post-translational modification of the N terminus is required for normal actin function.     

Coexistence of three major isoactins in a single sarcoma 180 cell

Actin in transformed sarcoma 180 cells is composed of the nonmuscle β and γ species and of a third, more acidic stable variant termed ζ. Two-dimensional peptide analysis shows that ζ is similar to β actin, differing in the mobility of only one tryptic peptide. Several lines of evidence indicate that ζ is not a modified β-actin species. This third actin species comprises 20% of the total labeled actin, has the same molecular weight as the β and γ actins and has a different mobility in isoelectric focusing gels from that of the known a actins from skeletal, cardiac and vascular smooth muscle. Like β and γ actin, ζ can be extracted with the actin depolymerizing factor from slime mold. Two-dimensional gel electrophoresis (isoelectric focusing) of the ³⁵S-methionine-labeled polypeptides synthesized by a single sarcoma 180 cell showed that all three major actin species coexist within the same cell. This analysis also showed for the first time the coexistence of α and β tubulin, vimentin, α actinin and three other polypeptides present in intermediate-filament-enriched cytoplast cytoskeletons (spots 12, 24 and 31). Determination of the ratio of γ plus β to ζ actin in different cytoskeletal preparations of intact and enucleated sarcoma 180 cells indicated that this actin species is not localized specifically to any of the major actin-containing structures preserved in the cytoskeletons.     

Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis

The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes.     

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis

Ca_V2.2 voltage-gated calcium ­channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with Ca_V2.2 in purified presynaptic terminal membranes. A number of known and new Ca_V2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-Ca_V2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct Ca_V2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of Ca_V2.2 was beyond reasonable doubt. The identification of the long-splice Ca_V2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.     

Chordate muscle actins differ distinctly from invertebrate muscle actins: The evolution of the different vertebrate muscle actins

A total of 30 actins from various chordate and invertebrate muscle sources were either characterized by full amino acid sequence data or typed by those partial sequences in the NH₂-terminal tryptic peptide which are known to be specific markers for different actin isoforms. The results show that most, if not all, invertebrate muscle actins are homologous to each other and to the isoforms recognized as vertebrate cytoplasmic actins. In contrast the actin forms typically found in muscle cells of warm-blooded vertebrates are noticeably different from invertebrate muscle actins and seem to have appeared in evolution already with the origin of chordates. During subsequent vertebrate evolution there has been a high degree of sequence conservation similar or stronger than that seen in histone H4. Urochordates, Cephalochordates and probably also Agnathes express only one type of muscle actin. Two types, a striated muscle-specific form and a smooth muscle form, are already observed in Chondrichthyes and Osteichthyes. Later in evolution, with the origin of reptiles, both muscle actins seem to have duplicated again; the striated muscle type branched into a skeletal- and cardiac-specific form, while the smooth muscle form duplicated into a vascular- and stomach-specific type. These findings support the hypothesis that each of the four muscle actins of warm-blooded vertebrates are coded for by a small number and possibly only one functional gene.     

NH2-terminal processing of Drosophila melanogaster actin. Sequential removal of two amino acids

Class II actins, such as Drosophila and mammalian skeletal muscle actins, have genes that code for a Met-X-Asp NH2 terminus where X is usually cysteine. These actins have an Ac-Asp NH2 terminus so two amino acids must be removed. To determine the nature of this processing, we labeled Drosophila Schneider L-2 cells with [35S]methionine or cysteine, isolated the actin, and analyzed the NH2-terminal actin tryptic peptides and their thermolysin digestion products. After a 4-h labeling period, we detected completed actin polypeptide chains with either an unblocked Asp or an Ac-Asp NH2 terminus. No intermediate with an NH2-terminal Cys or Met could be demonstrated. If, however, Drosophila mRNA was translated in a mRNA-dependent rabbit reticulocyte lysate system, an additional 43-kDa actin intermediate was observed. On the basis of thermolysin digestion studies and experiments using mild acid hydrolysis of a labeled actin NH2-terminal tryptic peptide fragment, we identified this intermediate as having an Ac-Cys-Asp NH2 terminus. In a time-dependent fashion, Ac-Cys was removed generating actin with an exposed NH2-terminal Asp which was subsequently acetylated to produce the mature form of actin. The removal of Met and the acetylation of Cys may occur early in translation while the nascent polypeptide chain is still attached to the ribosome. Subsequent processing occurs following completion of the synthesis of the actin polypeptide. The removal of Ac-Cys from Drosophila actin is thus similar to removal of Ac-Met from the NH2 terminus of class I actins although in the case of the class II actins, it is the second amino acid that is removed as an acetylated species.     

Disulfide-bonding between Drosophila laminin β and γ chains is essential for α chain to form αβγ trimer

Assembly of Drosophila laminin α, β and γ chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain-specific antibodies followed by two dimensional electrophoresis in which non-reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric β (or γ) with anti-γ (or -β) antibody revealed that β and γ form stable dimer before they are disulfide-bonded to each other. In contrast, α associates with neither monomeric β, monomeric γ nor βγ dimer without disulfide-bonding but only with disulfide-bonded βγ dimer to form αβγ trimers. These results thus demonstrated that the interchain disulfide-boding between β and γ is essential for α to form αβγ trimer. We also found that the αβγ trimer can be secreted with α chain either disulfide-bonded or not bonded to the disulfide-bonded βγ dimer.     

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins

Summary Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.Offprint requests to.: N. Mounier     

Alternate pathways for removal of the class II actin initiator methionine

Class II actin genes usually specify a polypeptide with a Met-Cys-Asp NH2 terminus, whereas the actin itself begins with an acetyl (Ac)-Asp(Glu). Previous studies with Drosophila actin showed that the first detectable intermediate is one with an Ac-Cys NH2 terminus which is subsequently cleaved in a novel reaction to expose the Asp. The initiator Met was probably removed early in translation as a free amino acid. To determine whether the class II actin initiating Met could also be removed in an acetylation-dependent manner, we translated Drosophila mRNA in a rabbit reticulocyte lysate in which protein acetylation was inhibited. After 60 min, three actin intermediates were detected, NH2-Met-Cys-Asp-actin, Ac-Met-Cys-Asp-actin, and NH2-Cys-Asp-actin. During processing in the presence of acetyl-CoA, three additional species were observed with NH2-terminal Ac-Cys-Asp, NH2-Asp, and Ac-Asp segments. In a time- and acetyl-CoA-dependent fashion, Met-Cys-Asp-actin was processed to the mature actin, presumably through an Ac-Met-Cys-Asp intermediate. Thus, two different pathways for removal of the initiator Met of class II actins, acetylation-dependent and independent, are possible. Since no class II actin intermediate containing the initiator Met is seen in vivo, although in class I actins this intermediate is observed, the most probable pathway for class II actins in vivo is the cotranslational removal of the initiator Met as a free amino acid.     

Soil restoration under pasture after topsoil removal: Some factors influencing C and N mineralization and measurements of microbial biomass

Factors influencing rates of C and N mineralization of soil and plant materials, and the reliability of different procedures for estimating microbial biomass, were measured in a soil (Typic Dystrochrept) that had been restored under grazed pasture in a temperate environment for 10–11 years after 20 cm of the original topsoil had been removed by stripping. Rates of net N mineralization were appreciably lower, but CO₂-C production higher, in the stripped than in the unstripped soil. These activities were not influenced directly by levels of soil mineral-N, but they were influenced by differences in plant composition. Herbage and litter, and roots, from the stripped plots were generally mineralized more readily to CO₂-C, but more slowly to net mineral-N, than were the corresponding materials from the unstripped plots. Rates of mineralization of herbage and litter, or roots, were mainly indistinguishable in stripped and unstripped soil, whereas rates of mineralization of all standing dead material were lower in stripped soil. Measurements of extractable-C flush, and of CO₂-C flush (using a fumigated soil control) and mineral-N flush by fumigation-incubation procedures, indicated that microbial biomass in stripped soil had recovered to at least 88 percent of the levels in unstripped soil. Substrate-induced respiration also generally indicated high levels of recovery of microbial biomass. The fumigation-incubation procedure appeared to under-estimate microbial biomass markedly in stripped soil when unfumigated soil controls were used; the used of a large soil inoculum (20 percent w/w) only sometimes overcame this problem. Possible reasons for apparent anomalies in estimation of microbial C are discussed.     

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2-3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A)+ RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.     

Tubulin and actin synthesis during brain development

Chick brain proteins from 5- through 13-day embryos were labeled with l-[³⁵S]methionine for 30 min in vitro and analyzed by two-dimensional gel electrophoresis. Autoradiographs of the gels were scanned with a computer-coupled densitometer to measure the relative rates of protein synthesis. The actins and the tubulins were the most abundant proteins and had the highest rates of synthesis. β and γ actin were synthesized at constant rates throughout this period of development, but the rate of tubulin synthesis increased fourfold. Six α tubulins and two β tubulins were distinguished, and they were all synthesized at all times. The relative rates of synthesis of these forms changed with development in a complex pattern, but the stoichiometry of α:β remained 1:1.     

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP

In pancreatic β-cells, K_ATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K_ATP channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K_ATP channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K_ATP channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K_ATP channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K_ATP channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.     

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP

In pancreatic β-cells, K_ATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K_ATP channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K_ATP channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K_ATP channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K_ATP channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K_ATP channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.     

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.     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.     

Mechanism of Deep-Sea Fish α-Actin Pressure Tolerance Investigated by Molecular Dynamics Simulations

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.     

Structural Comparisons of Muscle and Nonmuscle Actins Give Insights Into the Evolution of Their Functional Differences

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein. Received: 15 March 1996 / Accepted: 14 July 1996     

Purification of various forms of elongation factor 1 from rabbit reticulocytes

Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (α, β, γ, and δ). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1α, EF-βδ, EF-1βγδ) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1α is a single polypeptide of M_r 52,000, and has an isoelectric point of 9.1. EF-1βδ and EF-1βγδ are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1β, 5.5 for EF-1γ, and 4.8 for EF-1δ. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1α in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1βγδ is slightly more active than EF-1βδ. The similarity of the amino acid composition of EF-1γ and EF-1δ and the molar ratio of α:β:γ:δ in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1δ is probably a breakdown product of EF-1γ, and that the native form of EF-1H probably contains only the α, β, and γ subunits.