PubMed Health⌕ Search

Biomedical subjects

M L Greaser

Publications and source records attributed to M L Greaser.

At least 73 records · Page 4Linked to original sources
PubMed1985

Shortening velocity and myosin heavy chains of developing rabbit muscle fibers.

The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.

Aging↗
PubMed1985

Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition.

Extensive variations exist in the heavy and light chain components of myosin in vertebrate striated muscles. In the present study, we have characterized a specific contractile property, velocity of shortening, and protein subunit composition of single fibers from adult rabbit soleus muscles. Maximum velocity of shortening (Vmax) was measured using the slack test method, and the myosin composition of these same fibers was determined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. While most fibers were found to have velocities between 0.5 and 1.0 muscle length/s, several had velocities distributed between 1.33 and 2.99 muscle length/s. The fibers in the slower group had myosin subunits that were solely of the slow type; however, those in the faster group contained both fast and slow heavy chains and light chains. The velocity of shortening measured in fibers having both myosin types was highly correlated with the myosin heavy chain composition, with velocity increasing as the proportion of fast-type heavy chain increased. Variations in light chain composition, particularly fast and slow myosin light chain 1, appeared to occur independently of the variations in heavy chain composition, suggesting that some myosin molecules consist of mixtures of slow- and fast-type subunits.

Animals↗
PubMed1985

Immunocytochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils.

A monoclonal antibody specific to bovine cardiac titin has been identified. The antibody recognizes a common antigenic site in striated muscles of several species. In relaxed myofibrils, specific staining at the A-I junction resulted in a doublet of fluorescent bands within a sarcomere. The distance between the doublets in successive sarcomeres varied according to the degree of myofibrillar contraction. Staining on formamide-extracted myofibrils has confirmed that this epitope is located near the outer edges of isolated A bands. Selective extraction of myofibrillar proteins resulted in different staining patterns. Disrupting the structural integrity of the M-line or the A-band centre caused a significant amount of titin to translocate toward the Z-line region. In contrast, shortening of the A-band by removal of myosin from the ends of the thick filaments resulted in anti-titin staining moving closer to the M-line region. Several conclusions can be drawn from this study: (a) two aligned groups of titin molecules are placed symmetrically to the M-line in a sarcomere; (b) titin may attach directly or via intermediary protein(s) to sites near the M-line and Z-line such that the protein is under tension and (c) removal of proteins from either region results in titin staining in the opposite region. However, the edges of the A-band give some hindrance to collapse of the titin toward the M-line.

Animals↗
PubMed1985

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers.

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.

Animals↗
PubMed1984

Actin filaments in contracting preretinal membranes.

In 14 patients, preretinal membranes, causing retinal traction and severe visual impairment, were removed by vitrectomy and evaluated by light and electron microscopy using myosin subfragment-1 to stain actin filaments. Eight membranes were of vascular origin, six of nonvascular origin. All but one contained bundles of oriented actin filaments within a number of their nonvascular stroma cells, suggesting that the contractile protein action may have been involved in their clinically observed contraction.

Actins↗
PubMed1984

Iodination of myofibrils and myosin.

The relative reactivity of the tyrosine side chains in the proteins of skeletal muscle myofibrils was determined using iodination techniques. The destruction of ATPase activity of myofibrils and myosin by lactoperoxidase and chloramine-T iodination could be prevented by the attachment of cysteamine to the sulphydryl groups prior to the iodination reaction and subsequent regeneration with thioglycolate or dithiothreitol. Iodination using 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril did not require cysteamine treatment for retention of full enzymatic activity. The specific activity of the different proteins varied markedly with desmin, troponin-T, and tropomyosin having the highest labelling with all three iodination procedures. In contrast the myosin light chains had low specific activity when labelled in myofibrils or intact myosin. The isolated light chains, however, were much more highly iodinated. It appears that iodination may be a useful technique for examining protein-protein interactions in the myofibril.

Adenosine Triphosphatases↗
PubMed1984

Accumulation and localization of troponin-T in developing hearts of Ambystoma mexicanum.

Troponin-T (Tn-T) expression in developing hearts of axolotls, Ambystoma mexicanum, was studied with the use of polyclonal and monoclonal antibodies and SDS-polyacrylamide gel electrophoresis. In precontractile hearts (stage 32/33), Tn-T was present in addition to myosin, actin and tropomyosin as evidenced by the presence of the protein bands in SDS-gels and by indirect immunofluorescence. Tn-T was localized in amorphous collections at the peripheries of these precontractile cells. Hearts of normal and cardiac lethal mutant siblings were also analysed for Tn-T expression. No detectable differences in the quantity of protein present was observed by gel electrophoresis or by indirect immuno-fluorescence. The most striking difference concerned the localization of the protein. In normal hearts, Tn-T was primarily localized in the I-bands of organized myofibrils; however, in mutant cells the Tn-T was localized in amorphous collections at the cell peripheries suggesting a reduction of myofibrillar organization in these cells. No differences were observed in the contractile protein composition between normal and mutant embryonic hearts by gel electrophoresis experiments.

Actins↗
PubMed1983

Quantitative determination of myosin and actin in rabbit skeletal muscle.

The myosin and actin content of muscle tissue and purified myofibrils from rabbit psoas muscle has been determined. Myofibrils were purified using Percoll gradients, which allowed rapid separation from nuclei and connective tissue proteins. Myosin and actin were quantitated by amino acid analysis of the appropriate bands from sodium dodecyl sulfate/polyacrylamide gels. Muscle tissue contained 94 and 619 nmol/g wet weight of myosin and actin, respectively, while myofibrils had 0.82 and 5.37 mumol/g protein. Thus myosin contributed 43% and actin 22% of the myofibril protein mass. The value of 2.5 myosins per 14.3 nm repeat as calculated from these results suggests that thick filament models with mixtures of two and three crossbridges per repeat should be considered.

Actins↗
PubMed1983

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils.

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.

Actins↗
PubMed1983

Alterations in the Ca2+ sensitivity of tension development by single skeletal muscle fibers at stretched lengths.

The apparent length dependence in the calcium sensitivity of tension development in skeletal muscle has been investigated in the present study. At sarcomere lengths of 2.46-2.62 micron, the Hill plot of tension-pCa data is well fit by not one but two straight lines, suggesting the possible involvement of more than a single class of Ca2+-binding site in tension development. On the other hand, increasing the sarcomere length to 3.00-3.25 micron yielded Hill plots that were described by a single straight line, which indicates that at long lengths tension might be regulated by the binding of Ca2+ to a single class of Ca2+-binding sites, presumably the low affinity sites of TnC. This length-dependent transformation of the tension pCa relation occurred at free Mg2+ concentrations of both 0.05 and 3.2 mM. Although the mechanism of this effect is uncertain, plausible explanations for the biphasic Hill plot at the shorter lengths include the possible involvement of Ca2+ activation of the thick filaments and/or myosin LC2 phosphorylation in the process of tension development.

Animals↗
PubMed1983

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers.

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.

Animals↗
PubMed1982

Flexibility of myosin rod determined from dilute solution viscoelastic measurements.

The frequency dependencies of the storage and loss shear moduli, G' and G", of myosin rod solutions at 1.0 and 7.0 degrees C were measured by use of the Birnboim-Schrag multiple lumped resonator apparatus in solvents with and without glycerol. The infinite dilution moduli were determined and compared with theoretical models for a rigid rod and a freely jointed trinodular rod and with an empirical model for a semiflexible rod. Only the latter could fit the data. A rotational relaxation time of 25 mus and a slowest bending time of 3.1 mus, both reduced to water at 20 degrees C, were determined from the fit. A persistence length of about 130 nm was obtained from either the bending time, the rotational relaxation time, or the intrinsic viscosity. The average thermal excursion of the end of subfragment 2 was estimated to be 26 nm, more than sufficient to span the gap between the thick and thin filaments in muscles at all sarcomere lengths. Thus, a hinge between heavy meromyosin and light meromyosin does not appear necessary for myosin-actin contact. Young's modulus of about 1 x 10(9) N/m2 also makes it unlikely that subfragment 2 can be the elastic element in the Huxley-Simmons model of muscle contraction.

Animals↗
PubMed1982

Physiological effects accompanying the removal of myosin LC2 from skinned skeletal muscle fibers.

The possible role of the LC2 light chain of myosin in the contraction of vertebrate striated muscle has long been a subject of interest. This problem has been addressed in the present study in which the mechanical effects of partial removal of LC2 from skinned fibers of rabbit psoas muscle have been investigated. Each fiber was divided into three segments, thus allowing determinations of the LC2 content of the fiber 1) prior to extraction of the LC2 subunit, i.e. control, 2) following extraction of LC2, and 3) following readdition of LC2 to the fiber. Measurements of isometric tension and the maximum velocity of shortening were made in these fiber segments at each of the above stages of the extraction protocol. LC2 was partially removed from the fiber segments by treatment with a solution containing 20 mM EDTA, 50 mM KCl, 5 mM phosphate buffer, pH 7.0, for 120 min at 30 degrees C, a procedure modified from Chantler and Szent-Gyorgyi (Chantler, P. D., and Szent-Gyorgyi, A. G. (1980) J. Mol. Biol. 138, 473-492). LC2 content was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoretic techniques. The results indicate that removal of about one-third of the total LC2 within a fiber segment reduced Vmax by nearly 50%, with very little effect upon isometric tension. Readdition of LC2 to these fiber segments resulted in recovery of Vmax to near control values. These findings suggest that LC2 may modulate the kinetics of interaction of myosin with actin in mammalian skeletal muscle.

Animals↗
PubMed1981

Actin filaments in diabetic fibrovascular preretinal membrane.

A vitrectomy specimen from a diabetic patient was studied by light and electron microscopy using myosin subfragment 1 to decorate and identify actin filaments. The patient had proliferative diabetic retinopathy, a shrinking fibrovascular preretinal membrane associated with retraction of the thickened posterior hyaloid, and a localized traction retinal detachment. The fibrovascular tissue comprised normal mature collagen, few cells, and occasional blood vessels. The cells contained numerous thick bundles of the contractile protein, actin. We suggest that actin may have been involved in the contraction phenomena observed clinically and that membrane contraction might be blocked by pharmacologic treatment.

Actins↗
PubMed1980

X-ray diffraction studies of troponin-C crystals from rabbit and chicken skeletal muscles.

Crystals of troponin-C from rabbit and chicken muscles have been obtained using the vapor diffusion method. Crystals of rabbit troponin-C are tetragonal with a unit cell of a = b = 89.4 A, c = 79.9 A, space group P42, and 4 molecules/asymmetric unit. Diffraction from these crystals is limited to 7A and the crystals are unstable under x-ray irradiation, making them unsuitable for a high resolution structural determination. Crystals of chicken troponin-C are trigonal with a unit cell of a = b = 66.7 A, c = 60.8 A. The space group is P3121 or P3221 and there is only 1 molecule/asymmetric unit. These crystals diffract to at least 2.2 A, are very stable under x-ray irradiation, and are suitable for high resolution study.

Animals↗
PubMed1977

Determination of the amino acid sequence of troponon C from rabbit skeletal muscle.

The complete amino acid sequence of rabbit skeletal muscle troponin C (TnC) was determined from studies on overlapping peptides isolated from cyanogen bromide and tryptic digests. TnC was found to be a single polypeptide chain of 159 amino acid residues, including 2 residues of tyrosine and 1 each of cysteine, histidine, and proline. The amino end is acetylated, the calculated net charge at pH 7.0 is -29, and the calculated molecular weight is 17,965. There was no evidence for heterogeneity in the sequence. The previously proposed four apparent Ca2+ binding sites are located at residues 27 to 38, 63 to 74, 103 to 114, and 139 to 150.

Amino Acid Sequence↗