Michio Muguruma

Direct interactions between talin and actin

Talin was purified from chicken gizzard by a modification of the method of L. Molony et al. [J. Biol. Chem.(1987) 262, 7790-7795]. Unlike the talin purified by the previous method, the talin purified by the new method was found to bind to both F- and G-actin: Talin cosedimented with F-actin. On gel filtration of a mixture of talin and G-actin, a complex of talin and action was obtained. Talin stimulated the polymerization rate of G-actin. A major proteolytic fragment of talin that retained the binding ability to F-actin was also identified. These results indicate that talin can bind directly to actin and suggest that talin plays a key role in the organization of actin filaments at the actin-membrane attachment sites in vivo also.

Low-temperature osmotic dehydration improves the quality of intermediate moisture meats

Dehydration of meat at low temperature (4°C) with a new dehydrating sheet to obtain intermediate moisture meat produced more desirable results than did more rapid dehydration at 25°C, because the native state of meat proteins was better retained at the lower temperature. At low temperature with replacement of the dehydrating sheet once per day, the water content decreased to 57% after 150 h. Water activity (a(w)) dropped to 0·965. Myofibrils could be prepared readily from meat after 150 h at 4°C, and the myofibrils were induced to contract by addition of Mg(2+)-ATP solution and therefore retained biological activity. Electron microscopic observation showed that intermediate moisture meat prepared at 4°C retained the original muscle structure to a large extent. Myosin could be extracted easily from intermediate moisture meat dehydrated at 4°C after 10 days of storage at 25°C. No marked difference in the effect of dehydration on muscle was observed among species (beef, pork, chicken and rabbit). Results show that dehydration at low temperature (4°C) with use of the new dehydrating sheet enables intermediate moisture meat of good quality to be manufactured.

Humectants improve myosin extractability and water activity of raw, cured intermediate moisture meats.

Abstract Glycerol, propylene glycol and sorbitol were incorporated into salt-based intermediate moisture meats manufactured from porcine M. longissimus thoracis and bovine M. biceps femoris by dry curing and air drying at 4°C. Moisture content and water activity (aw) in cured pork were reduced by the addition of propylene glycol and sorbitol. Propylene glycol was more effective than sorbitol in lowering aw. The extractability of myosin heavy chain, used as an index of alteration of myofibrillar protein, decreased in intermediate moisture porcine meats with the addition of salt and was unaffected by sorbitol. However, use of glycerol and propylene glycol in cured and air-dried pork increased the extractability of myosin heavy chain. Whereas intact myofibrils could not be extracted from salt-cured, air-dried beef, myofibrils could be made from air-dried beef cured in the presence of 10% glycol, 5% propylene glycol and 4% sorbitol. Such myofibrils contracted immediately on addition of Mg2+-ATP. In addition, even after storafe for 5 months, including 30 days at 25°C, myosin heavy chain could be extracted from meat cured with this combination of humectants. In comparison with salt curing alone, curing meat with the above three humectants together, plus salt, results in intermediate moisture meats more like fresh meat.Glycerol, propylene glycol and sorbitol were incorporated into salt-based intermediate moisture meats manufactured from porcine M. longissimus thoracis and bovine M. biceps femoris by dry curing and air drying at 4°C. Moisture content and water activity (a(w)) in cured pork were reduced by the addition of propylene glycol and sorbitol. Propylene glycol was more effective than sorbitol in lowering a(w). The extractability of myosin heavy chain, used as an index of alteration of myofibrillar protein, decreased in intermediate moisture porcine meats with the addition of salt and was unaffected by sorbitol. However, use of glycerol and propylene glycol in cured and air-dried pork increased the extractability of myosin heavy chain. Whereas intact myofibrils could not be extracted from salt-cured, air-dried beef, myofibrils could be made from air-dried beef cured in the presence of 10% glycol, 5% propylene glycol and 4% sorbitol. Such myofibrils contracted immediately on addition of Mg(2+)-ATP. In addition, even after storafe for 5 months, including 30 days at 25°C, myosin heavy chain could be extracted from meat cured with this combination of humectants. In comparison with salt curing alone, curing meat with the above three humectants together, plus salt, results in intermediate moisture meats more like fresh meat.

Effect of calcium on extraction of Z-band proteins from I-Z-I brushes of rabbit striated muscle.

1. When rabbit striated muscle I-Z-I brushes were subjected to eleven extractions with three different extracting solutions, relatively more amount of proteins was extracted in the presence of 1 nM CaCl2 than in the presence of 5 mM EDTA or 5 mM ethyleneglycol-bisp(beta-aminoethylether)-N,N,N1,N1-tetra-acetic acid (EGTA). Among proteins extracted in the presence of 1 mM CaCl2, the protein components with molecular weights of 85,000, 95,000 and 220,000 were included, whereas these were not extracted in the other two. 2. Co-electrophoreses of 220,000 dalton protein and myosin heavy chain showed that these two protein components were distinct from each other. 3. Roles of Ca2+ are discussed on disintegration processes of I-Z-I brushes in special reference to its co-operative action with calcium-activated factor enzyme.

Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed.

Comparative studies on the extractability of collagen from aortas of stroke-prone spontaneously hypertensive and normotensive rats

The molecular states of collagen in the aortas of age-matched stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto rats (WKY) were studied by analyzing its extractability under defined conditions. The monomeric and oligomeric collagen extractable with 0.5 M acetic acid/6 M urea from aortic homogenates of 9-month-old SHRSP and WKY comprised approx. 0.6 and 2.0%, respectively, of the total collagen. On incubation of the acetic acid/urea-extracted residues with pepsin at 4 degrees C, the levels of the collagen alpha 1(I) and alpha 2(I) chains solubilized from the SHRSP residues were both less than 50% of those from the WKY residues. When the residues were incubated with pepsin at 15 or 25 degrees C, the differences became smaller. When the acetic acid/urea residues were hydrolyzed with cyanogen bromide, nearly identical peptide maps were obtained for SHRSP and WKY. The aortas from 2-month-old SHRSP and WKY contained much larger proportions of acid/urea-extractable collagen than those of the older rats (8.2 and 13% of the respective total collagen). The levels of the alpha 1(I) and alpha 2(I) chains solubilizable from the respective residues by pepsin at 4 degrees C were similar to each other. These results indicate that aortic collagen fibrils in SHRSP are stiffened more prominently than those in WKY.

Is there a protease that preferentially cleaves the M-line in partially dehydrated muscle?

When a partially dehydrated muscle fibre bundle (PDM, 65% H(2)O, pH 5.5, at 4 °C) was treated with a supernatant fraction (M-line-cleaving fraction: MCF) of muscle homogenate for 5 hr, the M-lines disappeared. MCF was extracted from rabbit skeletal muscles by homogenization with 15 mM HCl containing 0.5 M NaCl (pH 3.7), fractionated with 25-65% (NH(4))(2)SO(4) and clarified by Sephadex G-75. Rabbit psoas PDM was obtained with an osmotic dehydration sheet and glycerinated. One end of the bisected fibre bundle was incubated with 10 mM Na-acetate (pH 5.5), 1 mM EDTA, 5 mM β-mercaptoethanol (β-MCE), 150 mM KCl, 10 mM NaN(3) with MCF at 25 °C for 5 hr, the muscle being stretched and relaxed several times. The other end was incubated in the same solution, except that MCF was omitted (control). Electron microscopy showed the myofibrils broken down at the M-line in the presence of MCF. The myofilaments were closely packed near the Z-line and flared out at both ends near the centre of the sarcomere (bow-tie shape). Thus, the Z-line is not the only target of proteases and structural decomposition can also occur at the M-line under specific conditions. An M-line cleaving protease may exist in the MCF muscle extract.

Optical Measurements during Gelation Process of Muscle Protein under High Pressure

The dynamical properties of the pressure-induced gelation process of the muscle protein was investigated by observing the evolution of the turbidity spectra in the pressure-jump experiments. In the pressure-induced gelation process, time dependence of the slope of ln (τ d ) vs. ln λ (τ : turbidity, d : the thickness of the specimen and λ : wavelength) showed quite different behavior compared with the heat-induced one. Namely, as the time passed, the slope once decreased and then increased, suggesting that the depolymerization of F-actin takes place before the gelation process. Also, the surfaces of the pressure-treated products examined with a scanning electron microscope revealed the more smooth structure than that of the heat-induced gel.

Observation of Transmitted Light Spectra during Gelation Process of Actomyosin

The heat-induced gelation process of muscle protein, actomyosin, was investigated by observing the transmitted light spectra. First, the temperature characteristic of the gelation ( T g ′ ) was determined from the spectral changes, showing remarkable dependence on pH and [NaCl]. Moreover, the spectra varied drastically with time after the temperature-jump procedure. From the time dependence of the observed spectra, the turbidity was calculated to characterize the light-scattering properties. The logarithm of the turbidity showed approximately linear dependence on the logarithm of the wavelength, and the slope increased with time, suggesting the growth of the actomyosin assembly during the gelation process.

Gelation process of actomyosin

The heat‐, and pressure‐induced gelation process of actomyosin was investigated by observing the transmitted light spectra. First, pH and [NaCl] dependences of the characteristic temperature (T’g) related to the gelation were determined at atmospheric pressure. Then, the temperature variations of the transmitted light spectra were monotored after the temperature‐ and pressure‐jump procedures, revealing the growth of the actomyosin assembly during these gelation processes.The heat‐, and pressure‐induced gelation process of actomyosin was investigated by observing the transmitted light spectra. First, pH and [NaCl] dependences of the characteristic temperature (T’g) related to the gelation were determined at atmospheric pressure. Then, the temperature variations of the transmitted light spectra were monotored after the temperature‐ and pressure‐jump procedures, revealing the growth of the actomyosin assembly during these gelation processes.