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 and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha- An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha- actinin was present diffusely in the cytoplasm and on membrane- associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha- actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth- muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.     

Physico-chemical properties of rat and dog cardiac alpha-actinin

alpha-Actinin exists in several polymorphic forms which appear to be characteristic of the muscle type from which it is isolated. In order to determine the possible physiological role of this structural protein in cardiac muscle, we describe and compare here the physico-chemical properties of cardiac alpha-actinin from two different mammalian species, rat (fast contracting muscle) and dog (slow contracting muscle). Purification of cardiac alpha-actinin was achieved by chromatography on DEAE-cellulose and hydroxyapatite columns. The alpha-actinins isolated were different in their electrophoretic mobility (SDS-polyacrylamide gel electrophoresis), molecular size and alpha-helical content. However, their shape as revealed by electron microscopy and their activating effect on Mg2+-ATPase activity of actomyosin appear to be similar. These studies suggest that the rat and dog cardiac alpha-actinin are structurally different but functionally similar proteins.     

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.     

Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.     

Molecular evolution and structure of alpha-actinin

The N-terminal actin-binding domain of alpha-actinin is connected to the C-terminal EF-hands by a rod domain. Because of its ability to form dimers, alpha-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells. In the prototypic alpha-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats. We have found some atypical alpha-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one and two spectrin repeats, respectively. This implies that the four repeats present in modern alpha-actinins arose after two consecutive intragenic duplications from an alpha-actinin with a single repeat. Further, the evolutionary gene tree of alpha-actinins shows that the appearance of four distinct alpha-actinin isoforms may have occurred after the vertebrate-invertebrate split. The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early in the vertebrate radiation. The phylogeny also considers these atypical isoforms as the most basal to alpha-actinins of vertebrates and other eukaryotes. The analysis also positioned alpha-actinin of the fungi Encephalitozoo cuniculi close to the protozoa, supporting the suggestion that microsporidia are early eukaryotes. Because alpha-actinin is considered the basal member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits.

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Myosin heavy-chain isoforms in human smooth muscle

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Alpha-actinin from sea urchin eggs: biochemical properties, interaction with actin, and distribution in the cell during fertilization and cleavage

A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be approximately 95,000 on an SDS gel: it co-migrated with skeletal-muscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alpha-actinin. The appearance of sea urchin egg alpha-actinin as revealed by electron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. This protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha-actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration less than 1 microM. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.     

The relation between ATPASE activity and light chains of myosin in developing, adult and denervated muscles of several animals species.

Ca2+ATPase activity and light chains of myosin, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in developing, adult and denervated fast, slow and cardiac muscles of the rat, guinea-pig, cat, rabbit and chick were studied. It has been shown that in normal adult muscles the electrophoretic pattern of light chains of myosin reflects the myosin ATPase activity only when muscles from the same animal species are compared. In homologous muscles from adult animals differing in size, the size-dependent difference in myosin ATPase activity is not revealed in the electrophoretic pattern. Both in developing and in denervated muscle, changes in myosin ATPase activity are either connected with changes in the pattern of light chains of myosin or this pattern does not change. This relation is different in fast and slow muscles and also differs in chick and rabbit muscles. There are several possibilities of explaining the relation between ATPase activity of myosin and the pattern of light chains of myosin. The observation that myosin from the soleus muscle of 1-month-old rabbit contains light chains corresponding to both fast and slow type of myosin, indicates that the change in myosin ATPase activity during development is due to changes in the ratio between the fast and slow type of myosin.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Energy metabolism of carp swimming muscles

Summary Electromyography has been used to study the recruitment of red, pink and white muscle fibres of the Mirror carp at different swimming speeds. Locomotion below 0.3–0.5 L/S (lengths per second) is achieved primarily by fin movements after which the red myotomal muscle becomes active. Pink muscle fibres are the next type to be recruited at speeds around 1.1–1.5 L/S. White muscle is only used for fast cruising in excess of 2–2.5 L/S and during bursts of acceleration.Studies of the myofibrillar ATPase activities of these muscles have shown a ratio of 124 for the red, pink and white fibres respectively. The myosin low molecular weight components, which are characteristic of the myosin phenotype, have been investigated by SDS polyacrylamide electrophoresis. The light chain patterns of the pink and white muscles were identical and characteristic of the fast myosin phenotype. Red muscle myosin had a light chain pattern characteristic of slow muscles. It would appear that there is a relationship between the speed of contraction of the fibre types and the locomotory speed at which they are recruited.The activities of some enzymes of energy metabolism have also been determined in the three muscle types. Enzymes associated with oxidate metabolism have high, intermediate and low activities in the red, pink and white muscles respectively. Pyruvate kinase and lactate dehydrogenase activities were considerably higher in the pink than in either red or white muscles. It is suggested that the high capacity for anaerobic glycolysis of the pink muscle is associated with its recruitment for sustained effort at swimming speeds above which the fish can no longer meet all its energy requirements by gas exchange at the gills.Abbreviations used EDTA ethylenediamine tetraacetic acid - L/S lengths, sec^–1 - LDH Lactate dehydrogenase - PFK phosphofructokinase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

C-protein from rabbit soleus (red) muscle.

A new form of skeletal-muscle C-protein has been isolated from rabbit soleus (red) muscle. This new form of C-protein has been purified to homogeneity by a procedure similar to that used to purify C-protein from white skeletal muscle. In soleus muscle, only this new form of C-protein could be detected, whereas in psoas (white) muscle, only the previously identified form of C-protein was detected. The content of C-protein in rabbit soleus muscle is comparable with that found in psoas muscle. Other rabbit skeletal muscles composed of a mixture of fibre types contained at least two forms of C-protein. C-Protein derived from red skeletal muscle bound to myosin isolated from either red or white tissue, with maximum binding occurring at a ratio of approximately 13 microgram of red C-protein/100 microgram of myosin. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that C-protein isolated from red skeletal muscle has a molecular weight approx. 7% greater than that of C-protein isolated from white skeletal muscle. The amino acid content of both forms of C-protein was similar but major differences in the mol % of isoleucine and threonine were found. Antiserum against C-protein from white rabbit skeletal muscle formed a single precipitin line with rabbit C-protein on double in agar. This antiserum did not form a precipitin line when diffused against red C-protein from rabbit skeletal muscle. Also, this antiserum bound specifically to the A-band region of myofibrils isolated from psoas (white) muscle, but it did not bind to myofibrils prepared from soleus (red) muscle.     

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and alpha-actinin isoforms in developing rabbit skeletal muscle

In adult fast skeletal muscle, specific combinations of thin filament and Z-line protein isoforms are coexpressed. To determine whether the expression of these sets of proteins, designated the TnT1f, TnT2f, and TnT3f programs, is coordinated during development, we characterized the transitions in troponin T (TnT), tropomyosin (Tm), and alpha-actinin isoforms that occur in developing fetal and neonatal rabbit skeletal muscle. Two coordinated developmental transitions were identified, and a novel pattern of thin filament expression was found in fetal muscle. In fetal muscle, new TnT species--whose protein and immunochemical properties suggest that they are the products of a new TnT gene--are expressed in combination with beta 2 Tm and alpha-actinin1f/s. This pattern, which is found in both back and hindlimb muscles, is specific to fetal and early neonatal muscle. Just prior to birth, there is a transition from the fetal program to the isoforms that define the TnT3f program, TnT3f, and alpha beta Tm. Like the fetal program, expression of the TnT3f program appears to be a general feature of muscle development, because it occurs in a variety of fast muscles as well as in the slow muscle soleus. The transition to adult patterns of thin filament expression begins at the end of the first postnatal week. Based on studies of erector spinae, the isoforms comprising the TnT2f program, TnT2f, alpha 2 Tm, and alpha-actinin2f, appear and increase coordinately at this time. The transitions, first to the TnT3f program, and then to adult patterns of expression indicate that synthesis of the isoforms comprising each program is coordinated during muscle specialization and throughout muscle development. In addition, these observations point to a dual role for the TnT3f program, which is the major thin filament program in some adult muscles, but appears to bridge the transition from developmentally to physiologically regulated patterns of thin filament expression during the late fetal and early neonatal development.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.