Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. I. Mechanical response and ultrastructure.

The physiological properties of mechanical response and the ultrastructure in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia were studied to obtain information about excitation-contraction coupling in somatic smooth muscles responsible for smooth and slow body movement of molluscans. The contracture tension produced by 400 mM K was not affected by Mn ions (5--10 mM) and low pH (up to 4.0), but was reduced by procaine (2 mM). The K-contracture tension was not readily eliminated in a Ca-free solution containing ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). A large contracture tension was also produced by rapid cooling of the surrounding fluid from 20 degrees to 5 degrees--3 degrees C even when the preparation showed no mechanical response to 400 mM K after prolonged (more than 2 h) soaking in the Ca-free solution. These results indicate that the LBWM fibers contain a large amount of intracellularly stored Ca which can be effectively released by membrane depolarization. The fibers were connected with each other, forming the gap junctions, the desmosomes, and the intermediate junctions. The sarcoplasmic reticulum (SR) consisted of vesicular and tubular elements, and was mostly located near the fiber surface. The plasma membrane showed marked tubular invaginations of 600-800 A in diameter, with many branches (surface tubules), extending inwards for approximately 2 micron. These surface tubules were closely apposed to the SR, and the bridgelike structures analogous to those in the triadic junction of vertebrate skeletal muscle were observed in the space between the surface tubules and the SR. It is suggested that the influence of membrane depolarization is transmitted inwards along the surface tubules to cause the release of Ca from the SR.

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity.

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.

Basic properties of ATP-induced myosin head movement in hydrated myosin filaments, studied using the gas environmental chamber.

Although more than 50 years have passed since the monumental discovery of Huxley and Hanson that muscle contraction results from relative sliding between actin and myosin filaments, coupled with ATP hydrolysis, the mechanism underlying the filament sliding still remains to be a mystery. It is generally believed that the myofilament sliding is caused by cyclic attachment-detachment between myosin heads in myosin filaments and myosin-binding sites in actin filaments. Attempts to prove the myosin head movement using techniques of X-ray diffraction and chemical probes attached to myosin heads have failed to obtain clear results because of the asynchronous nature of myosin head movement. Using the gas environmental chamber (EC) attached to an electron microscope, we succeeded in recording myosin head movement in hydrated myosin filaments, coupled with ATP hydrolysis with the following results: (1)In the absence of actin filaments, myosin heads fluctuate around a definite neutral position, so that their time-averaged position remains unchanged; (2) On ATP application, myosin heads bind with ATP to be in the charged-up state, M-ADP-Pi, and perform a recovery stroke in the direction away from the myosin filament central bare zone and stay in the post-recovery stroke position; (3) In the actin-myosin filament mixture, myosin heads form rigor linkages with actin, and bind with applied ATP to be in the charged-up state, M-ADP-Pi, and perform a power stroke in the direction towards the myosin filament bare zone, while releasing ADP and Pi to stay in the post-power stroke position; (4) In both recovery and power strokes, myosin heads in the non charged-up state return to the neutral position. These results indicate that the charged-up myosin heads decide their direction of movement without being guided by actin filaments.

Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication.

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

Purification and characterization of a Ca(2+)-binding 450-kDa protein (MCBP-450) in the plasma membrane-enriched fraction from a molluscan smooth muscle.

In accordance with physiological and electronmicroscopic evidence that, in the anterior byssal retractor muscle (ABRM) of a common mussel Mytilus edulis, Ca2+ activating the contractile system is accumulated at the inner surface of the plasma membrane and at the membrane of sarcoplasmic reticulum (Ebashi, S. and Endo, M. (1968) Prog. Biophys. Mol. Biol. 18., 123-183; Suzuki, S. and Sugi, H. (1982) in The role of calcium in biological systems, Vol. I (Anghileri, L.J. and Tuffet-Anghileri, A.M., eds.), pp. 201-207, CRC Press, Boca Raton), we have found a high-molecular-mass (450 kDa) Ca(2+)-binding protein (MCBP-450) in the membrane fractions of the ABRM by 45Ca autoradiography of proteins transferred to nitrocellulose membrane (Rüegg, J. C. (1971) Physiol. Rev. 51, 201-248). MCBP-450, purified to electrophoretic homogeneity, exhibited Ca(2+)-dependent changes in mobility, tryptophan fluorescence, UV absorption and CD spectrum, indicating its Ca(2+)-dependent conformational changes. MCBP-450 has a high content of aspartic and glutamic acid (23.8%) and a high content of basic residues (27%). It has a high capacity Ca(2+)-binding site, which binds about 38 mol of Ca2+ per mol with an adissociation constant of 10(4) M-1, and a low-capacity Ca(2+)-binding site, which binds about 7 mol of Ca2+ per mol with an association constant of 10(5) M-1. These characteristics of MCBP-450 are consistent with the view that it is actually involved in regulating the contraction-relaxation cycle in the ABRM.

31P-NMR study of the regulation of glycogenolysis in living skeletal muscle.

Based on in vitro biochemical experiments, it is generally believed that glycogenolysis is regulated in two different ways; i.e., Ca2+ regulation at the phosphorylase step and phosphate-product regulation at the phosphofructokinase step. Recent studies on glycogenolysis in living vertebrate skeletal muscles using 31P nuclear magnetic resonance (NMR) presented evidence that glycogenolysis in vivo is regulated by Ca2+ released from the sarcoplasmic reticulum. We performed 31P-NMR studies on living frog skeletal muscle, and found that glycogenolysis is further regulated by the accumulation of phosphate products by contractile activity. Therefore, glycogenolysis in vivo can actually be regulated by the two mechanisms as predicted by in vitro biochemical studies.

In vitro ATP-dependent F-actin sliding on myosin is not influenced by substitution or removal of bound nucleotide.

To examine possible role of F-actin-bound nucleotide in ATP-dependent actin-myosin sliding, we prepared various actin filaments with different nucleotide contents and compared their sliding velocities on heavy mero-myosin in the presence of 2'-deoxyadenosine 5'-triphosphate (dATP) to exclude possible exchange of external ATP with the actin-bound nucleotide. Neither the sliding velocity nor the length of the actin filaments was significantly influenced by substitution or removal of actin-bound nucleotide, indicating that actin-bound nucleotide may not play a significant role in the sliding between actin and myosin.

Effect of inorganic phosphate and ADP on the myofilament sliding induced by laser flash photolysis of caged ATP.

Using the technique of laser flash photolysis of caged ATP, we have suggested that, under nearly isometric conditions, the unitary distance of myofilament sliding per ATP molecule (myosin head powerstroke) is about 10 nm. To give further information about the mechanism of myofilament sliding, we studied the effect of inorganic phosphate (Pi) and ADP on the photoreleased ATP-induced shortening of single glycerinated muscle fibers under very small external loads. Both the velocity and the distance of the myofilament sliding induced by 150 microM ATP increased by Pi (20 mM), and decreased by ADP (0.4 mM). On the other hand, Pi and ADP showed no significant effect on the myofilament sliding induced by 100 and 75 microM ATP. The potentiating effect of Pi on the myofilament sliding with 150 microM ATP can be explained as being due to the increase in population of AM.ADP.Pi with corresponding decrease in population of AM.ADP, and also the increase in population of M.ADP.Pi and M.ATP. Meanwhile, the inhibitory effect of ADP can be simply accounted for to be due to an accumulation of AM.ADP that already finished their force generating process. The ineffectiveness of Pi and ADP on the myofilament sliding with 100 and 75 microM ATP is consistent with the view that it is caused by almost synchronized single myosin head powerstrokes.

Effect of deuterium oxide on actomyosin motility in vitro.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.

Different cardiac myosin isoforms exhibit equal force-generating ability in vitro.

We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin.

Force-velocity relations of rat cardiac myosin isozymes sliding on algal cell actin cables in vitro.

The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers.

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.

The mode of transverse spread of contraction initiated by local activation in single crayfish muscle fibers.

Isolated single crayfish muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. The contraction initiated by a moderate depolarization spread inwards in a graded manner according to the magnitude and duration of depolarization. Increase of the depolarized area increased the distance of the inward spread for a given amount of depolarization. If a large area of the surface membrane was depolarized with a large pipette for a sufficiently long time, the contraction spread not only inwards, but further transversely passing through the center of the fiber. Successive brief depolarizations given at an appropriate interval could produce contraction more effectively for a given amount of total current than did a prolonged depolarization. On the other hand, the contraction initiated by a strong negative current was observed to spread around the whole perimeter but not through the center of the fiber. Each type of local contraction always spread along the striation pattern and not longitudinally. Possible mechanisms of these responses are discussed in connection with the transverse tubular system of the muscle fibers.

The mode of transverse spread of contraction initiated by local activation in single frog muscle fibers.

Isolated single frog muscle fibers were locally activated by applying negative current pulses to a pipette whose tip was in contact with the fiber surface. In contrast to the graded inward spread of contraction initiated by a moderate depolarization, the contraction in response to a strong negative current was observed to spread transversely around the whole perimeter but not through the center of the fiber. This response was elicited only with pipettes of more than 6 micro diameter. The response was still present if the sodium of the Ringer solution was replaced by choline, or the chloride was replaced by nitrate or propionate. The duration of the response appeared to be independent of the duration of stimulating current in fresh fibers, while the contraction lasted as long as the current went on in deteriorated fibers. The contraction was first initiated at the area of fiber surface covered by the pipette, and spread around the perimeter of the fiber with a velocity of 0.8-6 cm/sec. Possible mechanisms of the response are discussed in connection with the properties of the transverse tubular system, the possibility of some self-propagating process along the walls of the tubules being suggested.

X-ray diffraction and electron microscopy from Lethocerus flight muscle partially relaxed by adenylylimidodiphosphate and ethylene glycol.

The low-angle X-ray diffraction pattern from Lethocerus flight muscle fibres was recorded in rigor or under two conditions that modify crossbridge structure and behaviour, aqueous adenylylimidodiphosphate (AMPPNP) and AMPPNP + calcium in an ethylene glycol-water mixture. The effects on the 38.7 nm layer-line peaks (hk.6) of the diffraction patterns were studied in detail. In aqueous AMPPNP at room temperature, a condition in which rigor tension drops to half without loss of stiffness, the peaks remained nearly as intense as in rigor except for the 10.6, which dropped to half. In 20% (v/v) ethylene glycol-AMPPNP + 100 microM-Ca2+ at 23 degrees C (gly + pnp + Ca), a condition which removed muscle tension but left stiffness close to the rigor value, the 10.6 and 11.6 peaks greatly decreased but the 31.6 remained relatively high. The 14.5 nm meridional peak (00.16) became stronger on addition of AMPPNP and again on adding glycol + calcium. Considered in terms of constructively interfering filaments and crossbridges, the X-ray data indicated a transfer of diffracting crossbridge mass towards the thick filament as relaxation proceeds. We compared the X-ray diffraction patterns and crossbridge structure seen with electron microscopy (EM) under the same chemical conditions. EM and X-ray observations were mutually quite consistent overall. However, X-ray data indicated that more crossbridge mass was stereospecifically related to actin before fixation in the partially relaxed state (gly + pnp + Ca) than was suggested by the disordered crossbridge profiles seen by EM. We conclude that myosin heads at the start of the power stroke may both be closely related to their thick filament origins and form actin-determined attachments to the thin filament.

Targeting antigen-specific receptors on B lymphocytes to generate high yields of specific monoclonal antibodies directed against biologically active lower antigenic peptides within presenilin 1.

We describe a targeting technique that selects antigen-specific receptors on B lymphocytes using antigen driven selective production of monoclonal antibodies which are directed against functional peptide sequences within the presenilin 1 molecule that is believed to be related to the early-onset of familial Alzheimer's disease. Three different peptide sequences of presenilin 1 were constructed, one including the region around the amino acid position 300, where the putative cleavage site exists and the other two present in the N- and C-terminal regions of that site. The efficiency in production of the desired monoclonal antibodies was at least 5-40-fold that obtained with the poly(ethylene glycol) (PEG)-mediated method. In addition, monoclonal antibodies directed against each of the peptide sequence displayed a high specificity for the corresponding peptide, in contrast to the lack of success using the PEG method. Also, the selection of surface immunoglobulin receptors on B lymphocytes by the peptides of interest was confirmed by immunofluorescent analysis. Here we demonstrate that targeting B lymphocytes results in the successful and efficient production of highly specific monoclonal antibodies against the lower antigenic peptide sequences.

Studies on the synthesis of chemotherapeutics. 10. Synthesis and antitumor activity of N-acyl- and N-(alkoxycarbonyl)-5-fluorouracil derivatives.

A number of N-acyl and N-(alkoxycarbonyl)-5-fluorouracil derivatives possessing, for example, benzoyl, o-toluyl, acetyl, propionyl, heptanoyl, ethoxycarbonyl, phenoxycarbonyl, and benzyloxycarbonyl groups as N1 and/or N3 substituents were synthesized, and their antitumor activities were evaluated. The synthesis was achieved by a direct and two-step acylation of 5-fluorouracil and by selective N1-deacetylation of N1-acetyl-N3-substituted-5-fluorouracil under appropriate reaction conditions. Several N3-benzoyl- and N3-o-toluyl-5-fluorouracil derivates and showed significant activity against experimental tumor, and N1-acetyl-N3-o-toluyl-5-fluorouracil was found to be most promising among them. Further investigation revealed 12 to retain higher activity toward various tumors, with lower toxicity and good blood level, than either 1 or FT-207, even for oral administration.

Unitary distance of actin-myosin sliding studied using an in vitro force-movement assay system combined with ATP iontophoresis.

To obtain information about the mechanism of ATP-dependent actin-myosin sliding responsible for muscle contraction, we studied the "unitary" distance of sliding between a myosin-coated glass microneedle and actin filament arrays (actin cables) in a giant algal cell induced by iontophoretic application of ATP, attention being focused on the minimum distance of ATP-induced sliding when the amount of applied ATP was gradually decreased in the presence of hexokinase and D-glucose. The number of myosin heads interacting with actin cables was reduced to less than 100, as judged from the maximal force Po (approximately 100 pN) generated by myosin heads on the needle in the presence of 2 mM ATP. When the amount of iontophoretically applied ATP was decreased by reducing the amount of charge passed through the ATP electrode from 80 to 2 nC, the distance of ATP-induced actin-myosin sliding decreased almost linearly from approximately 100 to approximately 10 nm, no detectable actin-myosin sliding being observed with further reduction of the charge passed through the electrode. The amount of external load exerted by the bent microneedle was less than 1% of Po for the sliding distance < 50 nm. The actin-myosin sliding distances with a small amount of ATP slightly above the amount required to induce the minimum sliding distance were distributed around integral multiples of 10 nm, suggesting that the unitary distance of actin-myosin sliding coupled with ATP hydrolysis is of the order of 10 nm.

The effect of sarcomere length and stretching on the rate of ATP splitting in glycerinated rabbit psoas muscle fibers.

The effect of sarcomere length and stretching on the tension and the rate of ATP splitting was studied using small fiber bundles from glycerinated rabbit psoas muscle. The rate of ATP slitting was determined by measuring ADP production, while the tension development in response to a contracting solution (at pCa 5.3) was recorded in the same preparation. The isometric tension developed by the preparation decreased when the sarcomere length was increased. The decrease of tension development was accompanied by a decrease in the rate of ATP splitting. If a preparation exerting steady isometric tension was stretched by 5--10% at a velocity of 0.1 mm/s, the rate of ATP splitting was increased after stretching, while the steady isometric tension attained after stretching was also higher than the initial value. The extent of the excess ATP splitting caused by stretching decreased with increasing sarcomere length. These results suggest that the rate of the interaction cycle between actin and myosin molecules may increase as a result of stretching.

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers.

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).