Biomedical subjects

Y Maéda

Publications and source records attributed to Y Maéda.

At least 37 records · Page 2Linked to original sources

PubMed1995

Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions.

When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.

Amino Acid Sequence↗

PubMed1994

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.

Animals↗

PubMed1993

E. coli expression and characterization of a mutant troponin I with the three cysteine residues substituted.

A TnI cDNA was cloned from rabbit fast skeletal muscle, and site-directed mutagenesis was applied to replace all the three cysteine residues, Cys-48 and Cys-64 by Ala and Cys-133 by Ser. The mutant and wild-type TnI were expressed in E. coli and purified to homogeneity. No significant functional differences were observed between the mutant and the authentic TnI in terms of the interactions with TnT and TnC, and the ability of the reassembled Tn complex to regulate the acto-S1 ATPase activity in a calcium-dependent manner. These findings suggest that none of the cysteine residues in TnI are essential for the function of this protein and can be replaced to obtain a non-oxidizable mutant TnI which is much easier to handle and suitable as an alternative to the authentic TnI for various purposes, such as crystallization of TnI and the whole Tn, and 1H NMR studies.

Adenosine Triphosphatases↗

PubMed1993

Calcium ions and the structure of muscle actin filament. An X-ray diffraction study.

In order to investigate the effects of Ca(2+)-binding to troponin on the conformation of the muscle thin filament (consisting of actin, tropomyosin and troponin) in the absence of actomyosin interaction, two series of X-ray diffraction experiments were undertaken. Firstly, the small angle X-ray scattering from filaments in solution indicate the tropomyosin strands are centred at about 3.5 nm from the filament axis and this distance is calcium independent. Secondly, X-ray fibre diffraction patterns from the filaments orientated in glass capillaries were studied. The X-ray intensity of the 2nd actin layer-line increased in a highly co-operative manner at a concentration of free calcium ions [Ca2+] of 10(-6.8) M, which is the range in which muscle contraction is physiologically regulated. However, this intensity increase accounted for some 30% of the total increase observed in diffraction patterns from muscle on activation, suggesting that the Ca(2+)-binding alters the state of the thin filament, which then undergoes further changes upon interaction with myosin.

Actins↗

PubMed1993

A new crystal form of tropomyosin.

Tropomyosin crystals with a new morphology have been obtained from lobster tail muscle tropomyosin from which 11 residues at the carboxyl-terminus have been proteolytically removed to avoid head-to-tail polymerization. In contrast to the conventional Bailey crystal form in which the elongated tropomyosin molecules form a mesh, in the present crystals the molecules are packed side-to-side with the long axes parallel to the c-axis of the crystal. The unit cell is tetragonal with a = b = 109 A, c = 509 A, and the symmetry is either P4(1)2(1)2 or P4(3)2(1)2, with 4(1)(4(3)) helical axes parallel to the c-axis. This suggests that a group of molecules surrounding a local 4(1)(4(3)) axis is regarded as the building unit of the crystal. It is likely that the unit cell contains eight molecules with one molecule per asymmetric unit.

Amino Acid Sequence↗

PubMed1993

Cross-bridge attachment and stiffness during isotonic shortening of intact single muscle fibers.

Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.

Actins↗

PubMed1993

Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli.

Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC. beta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S1 ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials.

Amino Acid Sequence↗

PubMed1992

Isolation, purification and partial characterization of tropomyosin and troponin subunits from the lobster tail muscle.

In a search for an invertebrate muscle from which the muscle regulatory proteins could be obtained in a great quantity and at high homogeneity, the regulatory proteins, tropomyosin (Tm) and three subunits of troponin (Tn), have been isolated from the lobster tail muscle, purified and partially characterized. The calcium-sensitive ATPase of lobster myofibril was restored when purified lobster Tm and lobster Tn were added to actin. Quantitative SDS-polyacrylamide gel electrophoresis showed that the lobster muscle contains actin, Tm, Tn with a molar ratio 7:1:1 and that lobster Tn consists of three subunits, one of each I, C and T. Each subunit was identified according to its effect on the acto-S1 ATPase rate. The isomer composition in each fraction of purified Tn subunit and in Tm are different from the rabbit skeletal muscle proteins; Tm consists of a single species of polypeptide of M(r) 38,000; the TnT fraction appears to be homogeneous with M(r) 43,000; the TnI fraction contains five isomers, all showing similar isoelectric pH, differing in M(r) in the range from 28,000 to 31,000; two TnC fractions contain three isomers in total with a range of M(r) from 18,500 to 19,000. Further study of the lobster Tm elucidated that digestion by carboxypeptidase A gave rise to a homogeneous preparation of truncated and non-polymerizable Tm which is devoid of 11 residues at the C-terminus of the molecule. The C-terminal amino acid sequence of 11 residues is homologous to the thoracic isomer generated from Drosophila melanogaster Tm-I gene. The present study indicated that, despite heterogeneities owing to the occurrence of isomers, the lobster regulatory proteins serve as an invertebrate source of the proteins for structural and biophysical studies, alternative to vertebrate counterparts.

Actomyosin↗

PubMed1992

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.

Animals↗

PubMed1992

Expression in Escherichia coli and a functional study of a beta-troponin T 25 kDa fragment of rabbit skeletal muscle.

A 25 kDa fragment of beta-type troponin T (beta-TnT) was expressed in Escherichia coli, and its function as a component of the regulatory system for actomyosin ATPase was compared with that of the authentic counterpart, the full length alpha-TnT. The expressed species, designated as beta-TnT(N'-208), consists of 208 residues. It lacks the entire variable region at the amino-terminus and, near the carboxyl-terminus, a segment of 14 residues is changed from the alpha-type to the beta-type sequence. Functional tests indicated that the truncated beta-TnT was not distinguishable from the full length alpha-TnT, suggesting that neither deletion of the variable N-terminal region nor alteration of the type has a significant effect on the regulatory action of TnT.

Amino Acid Sequence↗

PubMed1991

C-terminal structure and mobility of rabbit skeletal muscle light meromyosin as studied by one- and two-dimensional 1H NMR spectroscopy and X-ray small-angle scattering.

Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy. X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin. The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile. The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other. Essentially the same pattern is observed in intact myosin. In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues. The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5. The pK values of the histidine residues vary between 5.7 and 7.0.

Amino Acid Sequence↗

PubMed1991

Rabbit skeletal muscle myosin. Unfolded carboxyl-terminus and its role in molecular assembly.

We have expressed in E. coli segments of the rod portion of rabbit skeletal fast muscle myosin and compared physical properties of two different species, LMM-30 and LMM-30C'. LMM-30 consists of 263 amino acids including the original C-terminus of myosin heavy chain. LMM-30C' is colinear with LMM-30, but is devoid of 17 residues at the C-terminus. 1H NMR spectroscopy indicates that the C-terminus of LMM-30, but not of LMM-30C' is unfolded and freely mobile. Furthermore, the present results show that the unfolded C-terminus is essential for molecular assembly of LMM-30; at pH 8.0 LMM-30, but not LMM-30C', formed aggregates upon decreasing the ionic strength.

Amino Acid Sequence↗

PubMed1991

Complete coding sequences of cDNAs of four variants of rabbit skeletal muscle troponin T.

Four variants of troponin T (TnT) cDNAs have been isolated and sequenced. These cDNAs have been derived from rabbit skeletal muscle, the most widely studied source of troponin, of a 11-day-old animal. One variant (TnT-1) contains the complete coding sequence, while in three variants the coding sequences are truncated at the 5' termini. The previously published amino acid sequence differs from the present cDNA-derived sequences at three locations. At least two, possibly all, of them are probably accounted for by errors in peptide sequencing. The present results are consistent with the two types of alternative splicing of TnT genes, both being first reported on the rat gene. (1) Highly variable sequences in the amino-terminal region are accounted for by the alternative splicing of exons 4-8 in an interchangeable but not mutually exclusive manner. (2) In the carboxyl-terminal region, the alternative splicing of two exons 17 (beta-type) or 16 (alpha-type) in mutually exclusive manner is consistent with the difference between all the four cDNAs, which express exon 17, and the previously published peptide sequence (derived from the adult muscle) in which exon 16 is present. This variation also corresponds to the finding in chicken skeletal muscle that the choice of exon 16 or 17 may be dependent on developmental stages. Finally, a sequence is observed corresponding to an extra exon or exons between exons 5 and 6. This sequence is shorter than that of the chicken skeletal muscle gene and is not detected in the rat skeletal muscle gene.

Amino Acid Sequence↗

PubMed1988

Transient kinetics and time-resolved X-ray diffraction studies in isolated single muscle fibres.

The timing of events associated with the contraction and relaxation of the force cycle is described in isolated single arthropod muscle fibers using the fluorescently labelled derivatives of the Ca2+ binding sub-unit of troponin TnC. The kinetics of the subtracted fluorescence (490-410 nm) response from injected TnCDANZ, labelled at the Ca2+ specific sites, shows a rapid rise which is some 90% complete at 50% force consistent with rapid Ca2+ binding to this sub-unit. Subsequently the TnCDANZ fluorescence decays 2x more slowly, at 12 degrees C, than force consistent with a slower release of this bound Ca2+. In fibers injected with both aequorin and TnCDANZ, the aequorin kinetics are essentially unaltered compared to control fibers in the presence of 10-100 microM TnCDANZ. The peak of the aequorin response occurs some 150-170 msec in front of the TnCDANZ peak and the T 1/2 for light decay is faster than either force or TnCDANZ decay, but there is a 'tail' to the aequorin light response (elevated free Ca2+) well into the relaxation phase, seen both in cannulated and intact muscle fibers. The kinetics of the fluorescence of TnCIAANS, labelled of the Ca2+-Mg2+ sites, shows a slow decrease (T 1/2 1.8 sec) and subsequent increase (T 1/2 2.5 sec) in fluorescence consistent with a slow loading and unloading of these sites with Ca2+ during a tetanus. Time resolved X-ray diffraction from intact muscle fibers indicate that forces of up to 600 kN/m2 can be developed at sarcomere lengths of 8-10 micron. Force shows a marked sarcomere dependency while the aequorin response is relatively insensitive. At these high forces, there is a marked change in intensity of the first actin layer line (A2 at 38 nm), consistent with S1 (cross-bridge) attachment, which has a T 1/2 for rise of 125-150 msec.

Actins↗

PubMed1988

Cause of changes in the thin filament-associated reflexions on activation of frog muscle--myosin binding or conformational change of actin.

Although the intensity increase of the 5.9 nm layer-line (5.9 L.L.) on activation can potentially be informative, systematic investigation is left to be done. The present study is aimed to assign the cause of the intensity increase. Using the synchrotron radiation, X-ray diffraction patterns were recorded from live sartorius or semitendinosus muscle of the frog (mostly at a temperature of 3-4 degrees C) either on the "Imaging Plates", a newly developed X-ray image recording system, or with the fast linear detector. The Imaging Plates enabled us to measure the disorientation of the thin filaments in the contracting muscle, so that the intensity profiles of the layer-lines were corrected for the disorientation. After the correction, a slight inward shift of the intensity peak of 5.9 L.L. was measured which is associated with the intensity increase on activation at 4 degrees C. At higher temperatures, 5.9 L.L. increases less. The extent of the intensity increase is linearly related to the sarcomere overlap, whereas the outer part of the 2nd actin layer-line (2nd L.L.) is as high as 60% at 10% overlap. The rise of the intensity of 5.9 L.L. is as fast as that of 2nd L.L., but much faster than the changes of the equatorial reflexions and the tension rise. Although these results do not let us assign the cause, some properties are elucidated of the structural change which causes the intensity increase.

Actins↗

PubMed1988

The time course of changes in the equatorial diffraction patterns from different muscle types on photolysis of caged-ATP.

Using the synchrotron X-ray source at DESY, Hamburg, we have measured the time courses of changes in the strongest equatorial reflections from small bundles of chemically skinned fibres from insect flight muscle, Limulus muscle and rabbit psoas and soleus muscles following the photolytic release of ATP. In all preparations the release of ca. 2 mM ATP caused the tension to relax with a complex time course, the final relaxation rates in insect and rabbit psoas fibres being ca. 10 times faster than those measured in rabbit soleus and Limulus fibres (ca. 50 ms cf. ca. 500 ms half times). However, in all fibre types there was a very rapid change in equatorial intensities towards relaxed values (half time less than 5 ms), the extent of this change and the occurrence of a slower phase of intensity change being dependent on the preparation. In insect the 1.0 and 2.0 intensities change rapidly to their relaxed values; in Limulus the 1.0 and 1.1 intensities change rapidly to within ca. 10% of their relaxed values; in rabbit psoas the initial, rapid 1.1 intensity fall is to within 30-40% of its relaxed value and is followed by a slower fall which appears to correlate with the rate of the final tension relaxation, e.g. phosphate ions accelerate both rates; in rabbit soleus the equatorial response is very similar to that of psoas fibres. These results are discussed in terms of the model of Goldman et al. in which a rapid ATP induced dissociation of rigor crossbridges is followed by the transient cooperative reattachment of some bridges which may proceed through at least part of the cross-bridge cycle.

Actomyosin↗

PubMed1986

Intensity increases of actin layer-lines on activation of the Limulus muscle.

Small angle x-ray diffraction patterns were recorded from isometrically contracting Limulus (horseshoe crab) telson levator muscle using a multiwire proportional-area detector on the storage ring DORIS. In the pattern a substantial increase in intensity is observed on the thin-filament-associated layer-line at 1/38 nm-1 (the first actin layer-line) with a maximum increase at a radial spacing of R = 0.07 nm-1 but there is a much smaller change in the intensity of the 5.9-nm layer-line, which also arises from the thin filament structure. The results suggest that during contraction the myosin heads, presumably being attached to the thin filaments, are arranged along the long-stranded helical tracks of the thin filaments but that the spatial relationship between the heads and the actin monomers varies. Intensity increases have also been observed (Maéda et al., manuscript in preparation) in the part of the patterns from frog muscle and barnacle muscle, which are attributable to the first actin layer-line. It is thus likely that the intensity increase of the first actin layer-line on the Limulus pattern is associated not with structural features which are special to Limulus muscle, but with the tension generating processes that are shared by muscles in general.

Actin Cytoskeleton↗

PubMed1985

X-ray diffraction study of fast and slow mammalian skeletal muscle in the live relaxed state.

Experimental methods have been developed by which fast and slow muscles of the mouse can be maintained in good condition for the time needed to record detailed X-ray patterns. The results presented here show that thick filaments of both types of fibre show only minor differences from those of frog muscle. However, in slow muscle alone a remarkable transformation of this structure can occur.

Animals↗