Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence.

Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band. Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin. The possible meaning of the different distribution of the two calcium binding proteins is discussed.     

Immunofluorescent subcellular localization of some muscle proteins: A comparison between tissue sections and isolated myofibrils

Summary The localization of parvalbumin in fish white muscle and of the calcium binding protein, of arginine kinase and of glycogen phosphorylase in crayfish tail muscle have been investigated by immunofluorescence using isolated myofibrils and muscle sections as starting materials.It is shown that the four proteins appear to be localized on the thin filaments when myofibrils are used as starting material. This result contrasts with previous observations where it appeared that parvalbumin in fish muscle and arginine kinase in crayfish muscle were distributed uniformly within the cell. This discrepancy is discussed in relation to the high solubility of these proteins.In the light of the present knowledge about striated muscles from these two organisms, it seems that the roles of parvalbumin in fish and of the calcium binding protein in crayfish are probably different.A preliminary report on this work was presented at the meeting of the Union of Swiss Societies for Experimental Biology, Zurich, 1977 (Benzonana et al., 1977a)     

Immunofluorescent subcellular localization of some muscle proteins: a comparison between tissue sections and isolated myofibrils.

The localization of parvalbumin in fish white muscle and of the calcium binding protein, of arginine kinase and of glycogen phosphorylase in crayfish tail muscle have been investigated by immunofluorescence using isolated myofibrils and muscle sections as starting materials. It is shown that the four proteins appear to be localized on the thin filaments when myofibrils are used as starting material. This result contrasts with previous observations where it appeared that parvalbumin in fish muscle and arginine kinase in crayfish muscle were distributed uniformly within the cell. This discrepancy is discussed in relation to the high solubility of these proteins. In the light of the present knowledge about striated muscles from these two organisms, it seems that the roles of parvalbumin in fish and of the calcium binding protein in crayfish are probably different.     

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.     

The dihydropyridine receptor: expression of 190 kD alpha 1 subunit in crayfish muscle.

The existence of dihydropyridine receptor in crayfish striated muscle was proved by Northern blot analysis and 3H PN 200--110 binding. The alpha 1 subunit is encoded by a 8300 nt mRNA population and is expressed as 190 kD protein in crayfish T-tubular system, which binds 3H PN 200--110 (Bmax 1.5 +/- 0.4 pmol/mg protein and KD 6.2 +/- 0.8 nmol/l). The purified protein is phosphorylated by cAMP-dependent protein kinase. The dihydropyridine receptor in crayfish striated muscle also contains alpha 2 subunit, which on Northern blot gives the same signal as the alpha 2 subunit from rabbit skeletal muscle.     

The regulation of muscle phosphorylase kinase by calcium ions,calmodulin and troponin-C

Although it has been believed for several years that calcium ions are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past three years that this concept has started to be placed on a firm molecular basis. It appears that the regulation of phosphorylase kinase $\text{in vivo}$ is achieved through the interaction of the enzyme with the two calcium binding proteins, calmodulin and troponin-C, and that the relative importance of these proteins depends on the degree of phosphorylation of the enzyme (figure 3). In the dephosphorylated form of the enzyme, troponin-C rather than calmodulin is the dominant calcium dependent regulator providing an attractive mechanism for coupling glycogenolysis and muscle contraction, since the same calcium binding protein activates both processes. On the other hand, the phosphorylated form of the enzyme can hardly be activated at all by troponin-C, although it is still completely dependent on calcium ions. Calmodulin (the δ - subunit) is therefore the dominant calcium dependent regulator of phosphorylase kinase in its hormonally activated state.

Recent work has demonstrated that phosphorylase kinase not only activates phosphorylase, but also phosphorylates glycogen synthase thereby decreasing its activity (45–49). The regulation of phosphorylase kinase by calcium ions may therefore also provide a mechanism for co-ordinating the rates of glycogenolysis and glycogen synthesis during muscle contraction.     

Subcellular distribution of ryanodine receptors in the cardiac muscle of carp (Cyprinus carpio)

We examined the subcellular localization of ryanodine receptors (RyR) in the cardiac muscle of carp using biochemical, immunohistochemical, and electron microscopic methods and compared it with those of rats and guinea pigs. To achieve this goal, an anti-RyR antibody was newly raised against a synthetic peptide corresponding to an amino acid sequence that was conserved among all sequenced RyRs. Western blot analysis using this antibody detected a single RyR band following the SDS-PAGE of sarcoplasmic reticulum (SR) membranes from carp atrium and ventricle as well as from mammalian hearts and skeletal muscles. The carp heart band had slightly greater mobility than those of mammalian hearts. Although immunohistochemical staining showed evident striations corresponding to the Z lines in longitudinal sections of mammalian hearts, clusters of punctate staining, in contrast, were distributed ubiquitously throughout carp atrium and ventricle. Electron microscopic images of the carp myocardium showed that the SR was observed largely as the subsarcolemmal cisternae and the reticular SR, suggesting that the RyR is localized in the junctional and corbular SR.     

p 34cdc2 kinase homologue in the preprophase band

Summary Immunofluorescence microscopy with a monoclonal antibody raised against the PSTAIR sequence, which corresponds to a peptide conserved in the p 34^cdc2 protein kinase throughout the phylogenetic scale including higher plants, was used to study the intracellular localization of p 34^cdc2 during the cell cycle in onion root tip cells. Although p 34^cdc2 was evenly distributed in the cytoplasm throughout the cell cycle, a more intense staining was observed in the cortical region, where the preprophase band of microtubules (MTs) was located. Double staining with the PSTAIR and plant tubulin antibodies showed that the width of p 34^cdc2 band was narrower than that of MT band. These data raise the interesting question regarding the possible role of p 34^cdc2 protein kinase in determining the division site in plant cells.     

Distribution of calcium during contraction and relaxation of crayfish skeletal muscle fibre.

A model of activation of muscle contraction has been applied to the crayfish isolated skeletal muscle fibre. The model is based on calcium diffusion and binding to specific regulatory sites in a sarcomere. Calcium ions activate interactions of contractile proteins and thus the generation of force. The model quantifies the relation between calcium released from intracellular stores and force elicited. Experimental tension records from isolated crayfish skeletal muscle fibres under voltage clamp conditions are analyzed. Model parameters were determined either via approximation of the onset of tension by the model solution or from the model based relations between the tension maximum, and depolarizing pulse length and amplitude. This allowed to determine time changes of free and bound calcium distribution in the sarcomere and the calcium release from terminal cisternae. The steady state calcium concentration at terminal cisternae showed S-shaped voltage dependence with saturation below approx. 10 mumol/l at positive membrane potentials.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Novel Reticular Calcium Binding Protein Is Purified on Taipoxin Columns

Abstract: We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute-phase proteins. Here we report the identification of the second protein as a 49-kDa lumenal calcium binding protein that we have named taipoxin-associated calcium binding protein 49 (TCBP-49). This protein contains six EF-hand putative calcium binding domains and the carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP-49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP-49 will allow direct tests of whether TCBP-49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.     

Activation of calcium/calmodulin regulated kinases.

Among numerous protein kinases found in mammalian cell systems there is a distinct subfamily of serine/threonine kinases that are regulated by calmodulin or other related activators in a calcium concentration dependent manner. Members of this family are involved in various cellular processes like cell proliferation and death, cell motility and metabolic pathways. In this contribution we shall review the available structural biology data on five members of this kinase family (calcium/calmodulin dependent kinase, twitchin kinase, titin kinase, phosphorylase kinase, myosin light chain kinase). As a common element, all these kinases contain a regulatory tail, which is C-terminal to their catalytic domain. The available 3D structures of two members, the serine/threonine kinases of the giant muscle proteins twitchin and titin in the autoinhibited conformation, show how this regulatory tail blocks their active sites. The structures suggest that activation of these kinases requires unblocking the active site from the C-terminal extension and conformational rearrangement of the active site loops. Small angle scattering data for myosin light chain kinase indicate a complete release of the C-terminal extension upon calcium/calmodulin binding. In addition, members of this family are regulated by diverse add-on mechanisms, including phosphorylation of residues within the activation segment or the P+1 loop as well as by additional regulatory subunits. The available structural data lead to the hypothesis of two different activation mechanisms upon binding to calcium sensitive proteins. In one model, the regulatory tail is entirely released ("fall-apart"). The alternative model ("looping-out") proposes a two-anchored release mechanism.     

Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C

The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles. These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.     

Lectin-based proteomic profiling of aged skeletal muscle: decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month- versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluorescein-conjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.     

Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium‐binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq‐like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin‐ and dynamin‐like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq‐like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium‐binding regions (EF hands) are conserved. The widespread occurrence of frq‐like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 165–175, 1999     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.     

Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study

Binding of calcium to calmodulin (CaM) causes a conformational change in this ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution. Complexes of calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the soluble nitroxide spin label, 4-hydroxyl-2,2,6, 6-tetramethylpiperidinyl-1-oxy, on the line widths and relaxation rates of the Met methyl resonances in samples of biosynthetically 13C-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+4-CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full exposure of the C-terminal Met residues and a partial exposure of the N-terminal Met side chains. Binding of the three target peptides blocks the access of the nitroxide surface probe to nearly all Met residues, although the mode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding.     

Autophosphorylating protein kinase activity in titin-like arthropod projectin.

The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.