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Biomedical subjects

T Wallimann

Publications and source records attributed to T Wallimann.

At least 145 records · Page 8Linked to original sources
PubMed1985

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography.

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.

Animals↗
PubMed1984

The interaction of skeletal myosin subfragment 1 with the polyanion, heparin.

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross-linking techniques and limited digestion studies. 1. The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly-(L-glutamic acid) causes a specific structural change in the 50-kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45-kDa species by a proteolytic cleavage restricted to its COOH-terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50-kDa----45-kDa conversion, suggesting that the S1-nucleotide interactions exhibit some resemblances to the polyanion-S1 binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45-kDa and 20-kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains. 2. Moreover, the binding of heparin to S1 resulted in the self-association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross-linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg-nucleotides. The interaction of cationic sites on S1 and possibly on the 50-kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F-actin abolished S1 aggregation.

Actins↗
PubMed1984

Function of M-line-bound creatine kinase as intramyofibrillar ATP regenerator at the receiving end of the phosphorylcreatine shuttle in muscle.

After 10 wash cycles, 0.8 u.e. of creatine kinase activity remained bound per mg of chicken pectoralis myofibrils which had been freed of soluble creatine kinase, mitochondria, and membranes. The bound creatine kinase is located at the M-band and contributes to the electron density of this sarcomeric structure (Wallimann, T., Pelloni, G.W., Turner, D.C., and Eppenberger, H. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4296-4300). By measuring the combined actin-activated Mg2+-ATPase and creatine kinase reactions of myofibrils by pH-stat, it was shown that the amount of M-line-bound creatine kinase activity was sufficient to rephosphorylate the ATP hydrolyzed in vitro by the actin-activated Mg2+-ATPase. The amount of M-line-bound creatine kinase and thus the ATP regeneration potential depended on the muscle type. It was higher in fast muscles and lower in slow muscles. Inhibition of myofibrillar creatine kinase or extraction of the M-line-bound enzyme abolished the ATP regeneration potential without affecting ATPase activity. Inhibitors of myokinase, mitochondrial ADP/ATP translocase, and respiration did not affect the ATP regeneration potential or the ATPase. M-line-bound creatine kinase, sufficient to support an ATP turnover rate of 6s-1 per myosin head, seems to have the capacity for the intramyofibrillar regeneration of most or all of the ATP hydrolyzed by the myofibrillar ATPase during muscle contraction. Thus, M-line-bound creatine kinase at the myofibrillar receiving end of the phosphorylcreatine shuttle is of physiological significance.

Actins↗
PubMed1984

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).

Animals↗
PubMed1984

Shadowing of elongated helical molecules (myosin, tropomyosin, collagen, and DNA) yields regular molecule-dependent heavy metal grain patterns.

Myosin and other alpha-helical molecules (tropomyosin, collagen) can now directly be adsorbed on EM support films, washed, air-dried, or frozen and freeze-dried. Using this method, the molecules were rotary or unidirectionally shadowed with different heavy metals (Pt/C, Ta/W, Ag) or with C alone. After shadowing at low elevation angles with Ta/W or Ag, myosin, tropomyosin, collagen, and DNA showed strikingly regular patterns of either single or coalesced heavy metal grains (bands) along their entire lengths. Even after shadowing with C alone, repetitive, granular accumulations or bands of C were found along the molecules. The different heavy metals and C displayed distinctive banding patterns on the molecules examined, all of which are characterized by different surface charge periodicities and pitch values. The patterns were quantified on the basis of the distances between grains or bands. Two most frequently measured distances between bands were found after shadowing with heavy metals. After shadowing with Ag the prevalent distances between grains were about twice as large as those after Ta/W shadowing. By evaporating a thin layer of carbon on the molecules before shadowing with heavy metals or by evaporating C alone (with no heavy metal) at 6 degrees, one of these two most prevalent distances between bands was attenuated or disappeared. It was demonstrated that the remaining most frequently measured distances between grains seemed to be related to relief periodicities, to the pitch of the double-coiled (myosin, tropomyosin) and triple-coiled alpha-helices (collagen) and fractions thereof. The attenuated distances between grains agreed very well with distances of periodic surface charges on the molecules examined. The investigation of the grain or band patterns showed that their characteristics appearance was molecule-dependent and caused both by periodic chemical (repeats of positive and negative surface charges) and periodic structural features (coiling of the helical strands). The examination confirmed the existence of periodic positive and negative surface charges along the myosin rod and suggested a value of about 17.0 nm for the hitherto undetermined pitch of the double-coiled myosin rod.

Animals↗
PubMed1983

Electron microscopy of scallop myosin. Location of regulatory light chains.

The heads of the Ca2+-sensitive myosin molecules from scallop muscle, contrasted for electron microscopy by rotary shadowing, display two appearances depending on the presence or absence of the regulatory light chains. The heads of intact myosin appear "pear-shaped" as described for vertebrate myosin (Elliott & Offer, 1978): they are widest at the end remote from the tail and taper to a narrower neck near their junction with the tail. In contrast, myosin heads that lack the regulatory light chains appear more globular. The neck region is no longer visible: the rounded heads appear directly attached to the tail or there is an apparent gap between the head and the tail. Two preparations of myosin subfragment-1 that differ in light chain content show a similar difference in appearance. Fab fragments of antibodies specific for the light chains bind to the myosin heads and can also be visualized in the electron microscope using rotary shadowing. Both Fab fragments specific for the regulatory light chains and Fab fragments specific for the essential light chains bind preferentially to intact scallop myosin in the narrow region of the myosin head near its junction with the tail.

Animals↗
PubMed1983

Light-chain movement and regulation in scallop myosin.

Photo-cross-linking techniques show that when scallop myosin or myofibrils are subjected to experimental conditions that cause relaxed muscles to go into rigor, the N-terminal portion of the regulatory light chain of myosin moves towards the essential light chain while the C-terminal portion stays in place. These changes occur on the myosin before combination with actin. Cross-linking of the N-terminal region to the essential light chain in rigor locks the myosin into a conformation such that calcium sensitivity of the actin-activated Mg-ATPase is lost.

Actins↗
PubMed1983

Isoenzyme-specific localization of M-line bound creatine kinase in myogenic cells.

Experiments using isolated fibre bundles or myofibrils of chicken skeletal muscle have shown that a relatively small portion of the muscle-specific MM-type of creatine kinase (CK) (EC 2.7.3.2) is specifically bound to the M-line and yet greatly contributes to the electron-dense M-line structure. Here we demonstrate the presence of M-line bound CK in cultured myogenic cells by removing the unbound sarcoplasmic CK through permeabilization with Triton X-100 and extensive washing of the cells prior to immunofluorescence staining. When stained with antibodies specific for M-CK subunits these cells exhibit bright fluorescence within the M-line region of myofibrils. Occasionally this cross-striated pattern is also observed in mononucleated presumably postmitotic myoblasts. Anti-B-CK incubation, in contrast, results in a weak, diffuse fluorescence at the Z-band. Even though these cells contain appreciable amounts of B-type CK, specific fluorescence at the M-line is never observed with anti-B-CK antibody thus ruling out the presence of BB-type or MB-type CK at this location. Therefore the presence of CK within the M-line structure of myogenic cells which contain all three CK isoenzymes seems to be restricted to the MM-type isoenzyme.

Animals↗
PubMed1983

Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes.

The nonreceptor, peripheral v proteins (Mr 43,000 proteins) are conspicuous components of the acetylcholine receptor-rich membranes and the Torpedo electrocyte, so far devoid of any known enzymatic function. Creatine kinase (adenosine 5'-triphosphate:creatine N-phosphotransferase, EC 2.7.3.2) is identified in distinct polypeptides belonging to the family of v proteins. Embryonic (70- to 90-mm embryos), neonatal, and adult electric organs of Torpedo marmorata contain two isoenzymes of creatine kinase: the BB (brain) and the MM (muscle) forms. The proportion of the two isoenzymes does not appear to change in the course of ontogenic and postnatal development. Only the BB isoenzyme appears to be associated with the acetylcholine-rich membranes in adult Torpedo. The creatine kinase can be purified to homogeneity by chromatographic procedures that exploit the richness in free sulfhydryl groups of the enzyme. Specific activities of 150 units/mg are obtained from electric tissue. The enzyme subunits identified by two-dimensional gel electrophoresis and immunoblotting techniques have pI values in the 6.0-6.5 region and apparent molecular weights in the 40,000-43,000 range, the latter values depending on redox conditions.

Animals↗
PubMed1983

Novel staining pattern of skeletal muscle M-lines upon incubation with antibodies against MM-creatine kinase.

Incubation of chicken skeletal muscle fibers with an excess of anti-M-creatine kinase (CK) immunoglobulin G and an excess of anti-M-CK Fab fragments leads to heavy decoration of the M-line (Wallimann, T., D.C. Turner, and H.M. Eppenberger, 1977, J. Cell Biol. 75:297-317) and to removal of the electron-dense M-line structure (Walliman, T., G. W. Pelloni, D.C. Turner, and H.M. Eppenberger, 1978, Proc. Natl. Acad. Sci. USA., 75:4296-4300), respectively. On the other hand, incubation with low concentrations of monovalent anti-M-CK Fab did not extract but rather decorated the M-line, giving rise to a distinct two-line staining pattern. A similar double-line staining pattern, although less pronounced, was also observed within the M-line of paraformaldehyde-prefixed myogenic cells, which after permeabilization were incubated with low concentrations of divalent anti-M-CK antibody. In both cases, the two decorated lines appearing in the middle of the A-band were spaced axially 42-44 nm apart and correspond most likely to the two M4 and M4' m-bridge rows described by Sjöström and Squire (1977, J. Mol. Biol., 109:49-68; 1977, J. Microscopy., 111:239-278). It is concluded that the muscle-specific form of creatine kinase, MM-CK, contributes mainly to the electron density of these M4 and M4' m-bridges within the M-line structure. This specific labeling pattern is a further demonstration that CK is an integral part of the M-line.

Animals↗
PubMed1983

Visualization of freeze-dried and shadowed myosin molecules immobilized on electron microscopic films.

The mica replication technique first described by Hall [5] has produced myosin molecules which were heterogeneous in appearance in terms of shadowing, decoration, contrast and background. Therefore, an alternative technique for the visualization of myosin molecules was developed: Myosin molecules are sprayed directly onto glow discharged or silicium-monoxide coated carbon filmed grids, omitting glycerol. After washing several times with distilled water, rapid freezing, and freeze-drying, the immobilized myosin molecules are visualized by shadow-casting at low temperature and at varying angles. After backing with carbon the "in situ" shadowed molecules are observed in the electron microscope. This technique has several advantages over the standard method in that it yields more reproducible results. It is potentially useful for investigating interactions of myosin binding proteins with myosin and for visualizing unshadowed myosin in the STEM.

Acetates↗
PubMed1981

An immunological approach to myosin light-chain function in thick filament linked regulation. 1. Characterization, specificity, and cross-reactivity of anti-scallop myosin heavy- and light-chain antibodies by competitive, solid-phase radioimmunoassay.

Antibodies specific for the regulatory light-chain (R-LC), "essential" light-chain (SH-LC), heavy-chain, and rod fragment of myosin from the striated adductor muscle of scallop (Aequipecten irradians) were prepared and characterized. A competitive, solid-phase radioimmunoassay on microtiter plates, a combination of two systems described earlier by Kuettner et al. [Kuettner, M. G., Wang, A. L., & Nisonoff, A. (1972) U. Exp. Med. 135, 579-595] and Klinman et al. [Klinman, N. R., Pickard, A. R., Sigal N. H., Gearhart, P. J., Metcalf, E. S., & Pierce, S. K. (1976) Ann. Immunol. (Paris) 127C, 489-502], was adapted and used for an immunological survey of different myosins and myosin light chains. Anti-myosin light-chain antibodies were specific for the homologous chain and did not cross-react with the heterologous one, i.e., regulatory and essential light chains of scallop myosin could be distinguished immunologically. These antibodies also had a high degree of species specificity. A partial cross-reactivity was obtained only for the light chains of two closely related molluscan species out of the over thirty invertebrate or vertebrates species tested. Two populations of anti-SH-LC antibodies were found which differed in their ability to abolish regulation of scallop myofibrils and also in their immunological reactivity with cyanogen bromide fragments of teh SH-LC. A comparison of the cross-reactivity of the intact SH-LC with its CNBr fragments showed that most antigenic sites of the SH-LC were available to the antibodies. Free light chains and light chains associated with myosin reacted with antibodies in a very similar manner, indicating that the association of the light chains with myosin may not be accompanied by major conformational changes. Antibodies against scallop myosin heavy chain and rod fragment cross-reacted to a variable extent with all invertebrate myosins but with none of the vertebrates species tested. The antibodies did not cross-react with platelet and Physarum myosins. The heavy and light chains of myosin from scallop striated adductor, mantle, and foot were found toi be immunologically identical, whereas myosin from smooth adductor showed some differences mainly in the heavy-chain portion which forms the subfragment-l region of the myosin molecule. Heavy and light chains of scallop heart muscle myosin differed significantly from those of striated adductor muscle. Cross-reactivity did not depend on the regulatory properties of myosin.

Animals↗
PubMed1981

An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain.

Specific antibodies directed against the regulatory light chains (R-LC) or essential light chains (SH-LC) of scallop myosin abolished calcium regulation in myofibrils, myosin, and heavy meromyosin by elevating the actin-activated Mg2+-ATPase activity in the absence of calcium. Calcium dependence was completely eliminated at molar ratios of 2.5-3 antibodies bound per myosin. Monovalent anti-R-LC Fab and anti-SH-LC Fab fragments also desensitized myofibrils fully. High Ca2+-ATPase activity remained unaffected by the antibodies. Anti-SH-LC IgG reduced to about one-half the actin-activated Mg2+-ATPase in the presence of calcium and the potassium-activated ethylenediaminetetraacetic acid (EDTA)-ATPase activities. Anti-SH-LC Fab, however, desensitized without inhibiting the actin-activated Mg2+-ATPase. The desensitizing effect of both antibodies was abolished by prior absorption with the homologous myosin light chain. Calcium binding and R-LC and anti-SH-LC IgG's and by anti-SH-LC Fab. The anti-R-LC Fab fragment induced a significant (70%) dissociation of R-LC from myofibrils and myosins with concomitant losses in calcium binding. In contrast, anti-R-LC IgG prevented the dissociation of R-LC from myosin by EDTA. Binding of anti-R-LC IgG to myofibrils was proportional to thier R-LC content. Increased amounts of anti-SH-LC IgG were bound by myofibrils devoid of R-LC. Bound anti-SH-LC antibody significantly inhibited the reuptake of R-LC by EDTA-treated myofibrils as well as the full binding of anti-R-LC antibody. Certain rabbits produced a population of anti-SH-LC antibodies which were specific for this light chain and bound extensively to myosin but failed to desensitize it (nondesensitizing anti-SH-LC antibody). The desensitizing and nondesensitizing anti-SH-LC populations bound to different regions of the SH-LC on the myosin, and the binding of the two types of antibody to the SH-LC was nearly additive. The nondesensitizing SH-antibody inhibited the reuptake of R-LC less, and its binding to myofibrils was not influenced by the absence of R-LC. These studies indicate a direct or indirect involvement of the SH-LC's in myosin-linked regulation, raise the possibility of an interaction between the R-LC and SH-LC, and confirm the regulatory function of the scallop R-LC. A model for a relative location of the two types of light chains and the involvement of the subfragment-2 region of myosin linked regulation is discussed.

Adenosine Triphosphatases↗