S. Victor Perry

Polymorphic forms of troponin T and troponin C and their localization in striated muscle cell types.

Abstract 1. 1. Immunochemical studies have shown that the major forms of troponin T present in fast skeletal, slow skeletal and cardiac muscles are different proteins. 2. 2. Similar studies indicate that the major form of troponin C present in fast skeletal muscles differs from troponin C present in slow skeletal and cardiac muscle cells. The forms of troponin C present in slow skeletal and cardiac muscles are immunochemically very similar. 3. 3. The antibodies to the polymorphic forms of troponin T and troponin C are specific for the muscle type, except in the case of the slow skeletal and cardiac muscle forms of troponin C. 4. 4. By the immunoperoxidase technique, it has been shown that the fast skeletal muscle troponin T is localized in type II cells and slow skeletal muscle troponin T in type I cells. 5. 5. Fast skeletal muscle troponin C is present in type II cells and a different troponin C, identified by its reaction with the antibody against cardiac troponin C, is present in type I cells. 6. 6. It is concluded that in normal adult skeletal muscle, fast muscle forms of troponin I, troponin T and troponin C are present together as a homocomplex in type II cells and the slow muscle forms exist as an analagous homocomplex in type I cells.

THE AMINO ACID SEQUENCE OF THE TROPONIN C-LIKE PROTEIN (MODULATOR PROTEIN) FROM BOVINE UTERUS

Recent studies have shown that a troponin C-like protein is present in vertebrate smooth muscle [l-5] . This protein can form Ca”-dependent complexes with troponin I from fast skeletal muscle in a similar manner to those obtained with skeletal muscle troponin C [2,3] . It can be isolated from rabbit uterus using an affinity chromatographic procedure which depends on this property of complexing with skeletal muscle troponin I [l] but amore convenient, relatively simple, method of isolating the protein from smooth muscle and other tissues in good yields has recently been described [3]. The troponin C-like protein from smooth muscle possesses other properties similar to those of skeletal muscle troponin C. These are the abilities to inhibit the phosphorylation of troponin I by 3’,5’-cyclic AMP-dependent protein kinase and to neutralise the inhibitory activity of skeletal troponin I on the Mg2+-stimulated ATPase of desensitized skeletal actomyosin [2] . Unlike skeletal muscle troponin C, however, the smooth muscle protein activates cyclic nucleotide phosphodiesterase and contains the unusual amino acid, trimethyl lysine [3] . These latter properties are a feature of the Ca2+binding protein of bovine brain, the brain modulator protein, which Amphlett et al. [6] have demonstrated can substitute in the skeletal troponin complex in the place of troponin C and will restore full calcium sensitivity to desensitized actomyosin in the presence of troponin I and tropomyosin alone. Cheung et al. [7] have shown that a protein similar to the brain modulator protein is present in many of the tissues of mammals, and Waisman et al. [8] have

The localization of the different forms of troponin I in skeletal and cardiac muscle cells

Abstract 1. 1. Antibodies raised against troponin I isolated from human cardiac and rabbit fast and slow skeletal muscles have been shown to be specific for the polymorphic forms of troponin I against which they were raised, i.e. they are tissue specific. 2. 2. These antibodies reacted with the polymorphic forms of troponin I, against which they were raised, that are present in tissues of other species such as the rhesus monkey, hamster and rat, i.e. they were species non-specific. 3. 3. Using the immunoperoxidase staining technique it has been shown that the fast and slow forms of troponin I are located in different cells in virtually all adult normal muscles examined. 4. 4. By comparison of the ATPase staining of skeletal muscle sections at pH 9.4 and 4.2 it is concluded that the fast form of troponin I is located in type II fibres and the slow form in type I fibres. 5. 5. It is suggested that immunoperoxidase staining with the antibodies to the fast and slow forms of troponin I provides an unambiguous new method of muscle fibre typing.

The effect of denervation on the distribution of the polymorphic forms of troponin components in fast and slow muscles of the adult rat

SummaryIn the soleus muscle of the normal rat the number of cells containing fast troponin I decreased and those containing slow troponin I increased after birth until less than 10% stained for the fast form in the adult muscle. On denervation of soleus muscle this pattern of change was reversed with the result that the majority of cells stained for fast troponin I. The change was more rapid when denervation was carried out at 12 weeks rather than at 52 weeks of age. Denervation of extensor digitorum longus and tibialis anterior muscles produced little change in the distribution of fast and slow troponin I over a period of 12 weeks. After long periods (>24 weeks) of denervation of these fast muscles, fast troponin I was observed in cells in which originally only slow troponin I could be detected. Similar results to those obtained with troponin I in both fast and slow muscles were obtained using antibodies to the fast and slow forms of troponin C and troponin T.

Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin-I: structure-activity implications of ordered phosphorylation

We have used NMR spectroscopy to monitor the phosphorylation of a peptide corresponding to the N‐terminal region of human cardiac troponin‐I (residues 17–30), encompassing the two adjacent serine residues of the dual phosphorylation site. An ordered incorporation of phosphate catalysed by PKA was observed, with phosphorylation of Ser‐24 preceding that of Ser‐23. Diphosphorylation induced a conformational transition in this region, involving the specific association of the Arg‐22 and Ser‐24P side‐chains, and maximally stabilised when both phosphoserines were in the di‐anionic form. The results suggest that the second phosphorylation at Ser‐23 of cardiac troponin‐I is of particular significance in the mechanism by which adrenaline regulates the calcium sensitivity of the myofibrillar actomyosin Mg‐ATPase.

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.

Two forms of the P light chain of myosin in rabbit and bovine hearts

Heavy and light chains of myosin exist in a number of forms of different primary structure, various combinations of which are responsible for the large number of isoenzymic forms of the molecule present in muscle [ 1-11 ]. The precise combination of these forms of the components of the myosin molecule is characteristic of the muscle type and its stage of development. Changes in the isoenzymic composition of myosin can be observed, for example, after exposure to hormones such as thyroxine [12,13], after changes in innervation [14,15] and after chronic overloading in the heart [ 16]. Several forms of the alkali light chains are often present in a given skeletal muscle cell type [17,18]. We have demonstrated that the P light chain exists in two forms, P1 and P2, in ventricular muscle from several species [ 19]. This observation is supported by the recent report of two P light chains of different amino acid sequence in chicken ventricular myosin [20]. We now present evidence that the two forms of P light chain of myosin in bovine ventricle also have a different sequence and that the relative amounts of the two P light chains change during development in the rabbit heart.

The Regulatory Effects of Tropomyosin and Troponin-I on the Interaction of Myosin Loop Regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

Effect of thyroidectomy on the distribution of the fast and slow forms of troponin I in rat soleus muscle

The effects of thyroid hormones on the properties of muscle in both experimental and clinical situations are well-documented [1-9]. These studies indicate that the speeds of contraction and relaxation in skeletal muscle are related to the thyroid hormone status and that the changes in physiological properties of the muscles observed when this status is altered are associated with appropriate changes in the proportion of type I and type II fibres [6,7,9,10]. The modifications that occur in the myosin light chain composition after thyroidectomy correlate well with the changes in fibre types and physiological properties [8,10]. As similar changes in myosin light chain composition can be induced by cross innervation and by chronic electrical stimulation, some workers have concluded that the thyroid effect on skeletal muscle may be neurally mediated [8]. On the other hand, the results in [11] suggest that thyroid hormones may have a direct effect on gene expression at least so far as mitochondrial enzymes are concerned. Many of the studies on thyroid effects have been carried out on rat soleus muscle without making allowance for the pronounced changes in the proportion of type I and type II fibres which in the normal animal take place for at least up to 24 weeks after birth [ 12,13]. We have, therefore, re-examined the effect of thyroidectomy on fibre type changes in the rat soleus using the immunoperoxidase procedure with antibodies to the fast and slow forms of troponin I to identify cell types and paying particular attention to the continuing maturation process that occurs in this muscle. Our studies suggest that thyroid hormones

Characterization and fibre type distribution of a new myofibrillar protein of molecular weight 32 kDa

SummaryA new basic protein of molecular weight 32 kDa has been isolated and purified to homogeneity from skeletal muscles rich in type I fibres. By the use of a specific monoclonal antibody, the protein has been shown to be present in all type I fibres and some type II fibres, the number of which varies with the muscle and the region of the muscle sectioned. A protein of similar properties could not be isolated from rabbit muscles consisting predominantly of type II fibres. By fluorescence microscopy, the protein has been shown to be located in the Z-disc from which the presence of divalent cations, probably calcium, facilitates its extraction at low ionic strength. The protein is unusual in that its distribution does not correlate completely with the known muscle fibre types and in that as yet there is no evidence for the presence of an isoform in those cells that do not stain with the specific antibody for the 32 kDa protein isolated from slow muscles.