Biomedical subjects

Hiroyuki Iwamoto

Publications and source records attributed to Hiroyuki Iwamoto.

22 records · Page 2Linked to original sources

PubMed2003

High mechanical efficiency of the cross-bridge powerstroke in skeletal muscle.

We were interested to estimate the maximum mechanical efficiency with which chemical energy derived from ATP hydrolysis is converted into mechanical work by individual cross-bridges when they perform their powerstroke synchronously. Glycerinated rabbit psoas muscle fibres, containing ATP molecules almost equal in number to the cross-bridges within the fibre, were activated to shorten under various afterloads by laser-flash photolysis of caged Ca(2+). In these conditions, almost all the cross-bridges are in the state where the ATP is hydrolyzed but the products have not yet been released from the cross-bridge (M-ADP-P(i)) immediately before activation, and can hydrolyze only one ATP molecule during the flash-induced mechanical response. Power output records of the fibres following activation indicated that the cross-bridges actually started their powerstroke almost synchronously. The amount of ATP utilized at 1 s after activation was estimated from the amount of isometric force developed after interruption of fibre shortening, while the amount of work done was calculated by multiplying the amount of afterload by the distance of fibre shortening. A conservative estimation of the maximum mechanical efficiency at a load of 0.5-0.6 P(o) was 0.7, suggesting that the actual maximum mechanical efficiency of cross-bridge powerstrokes may be close to unity.

Adenosine Triphosphate↗

PubMed2002

States of thin filament regulatory proteins as revealed by combined cross-linking/X-ray diffraction techniques.

The regulatory protein system in the skeletal muscle thin filaments is known to exhibit three discrete states, called "off" or "blocked" (no Ca2+), "on" or "closed" (with Ca2+ alone) and "potentiated" or "open" (with strongly bound myosin head) states. Biochemical studies have shown that only weak interactions with myosin are allowed in the second state. Characterization of each state is often difficult, because the equilibria among these states are readily shifted by experimental conditions. To overcome this problem, we chemically cross-linked the skeletal muscle thin filament in the three states with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), in overstretched muscle fibers. The state of the regulatory proteins was monitored by measuring the intensity of the second actin layer-line (2nd LL) reflection in X-ray diffraction patterns. Structurally, the thin filaments cross-linked in the three states exhibited three corresponding discrete levels of 2nd LL intensities, which were not Ca2+-sensitive any more. Functionally, the thin filament cross-linked in the "off-blocked" state inhibited strong interaction with myosin head (subgfragment-1 or S1). The thin filament cross-linked in the "potentiated-open" state allowed strong interaction and full ATPase activity of S1 as described previously. The thin filament cross-linked in the "on-closed" state allowed strong interactions with S1 and actin-activated ATPase without enhancing the 2nd LL to the level of "potentiated-open" state, contrary to the expectations from the biochemical studies. The results demonstrate the potential of EDC as a tool for studying the states of calcium regulation, and the apparent uncoupling between the 2nd LL intensity and the function provides a new insight into the mechanism of thin filament regulation.

Actins↗

PubMed2002

Direct x-ray observation of a single hexagonal myofilament lattice in native myofibrils of striated muscle.

A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of approximately 2.5-microm-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.

Animals↗

PubMed2002

A lipase-inhibiting protein from lipoxygenase-deficient soybean seeds.

A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of beta-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498-1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified beta-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a beta-amylase, in soybean seed.

Amino Acid Sequence↗