Structural Changes in Titin and Nebulin Filaments Specific to Calcium Ions at 0.1 mM: Factors of Meat Tenderization During Postmortem Aging

ABSTRACT Postmortem structural changes in titin and nebulin filaments were investigated by incubating isolated myofibrils in a solution containing 0.1 mM calcium ions and various concentrations of a protease inhibitor. The inhibition curves showed 2 abnormal steps with increases in the concentration of leupeptin or calpastatin domain I. While the amounts of unchanged titin and nebulin were constant in the 1st step, the 2nd occurred at higher protease inhibitor concentrations. These facts indicated that excess amounts of leupeptin and calpastatin domain I caused deterioration in titin and nebulin properties, thus interfering with the binding of calcium ions. We concluded that the severance of titin and nebulin filaments in the 1st step were induced by calcium ions at 0.1 mM.     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

Carp Natural Actomyosin: Thermal Denaturation Mechanism

Structural changes of actomyosin, the major protein of muscle, on heating have been estimated on ATPase activity. We investigated carp actomyosin molecule changes on heating based on biophysical and biochemical techniques. Actomyosin molecules began to unfold at ～30°C. Hydrophobic amino acid residues and SH groups, which had been inside the molecule, emerged to the surface. Because of hydrophobic interactions and disulfide bonds, actomyosin molecules formed aggregates. At > 40°C, a part of myosin molecules was dissociated from actin filaments. Thus, dissociated myosin and the myosin-lacking molecules co-existed. In addition, fragmentation of actin filaments was observed, which was associated with the dissociation of myosin molecules. At ≥ 60 °C actomyosin molecules formed larger aggregates, in which no filamentous shape was observed. This aggregation occurred mainly by formation of SS bonds.     

Contribution of major structural changes in myofibrils to rabbit meat tenderisation during ageing

The contribution of major structural (myofibrillar fragmentation upon mechanical treatment) and ultra-structural (Z-line degradation, loss of electron density of M-line, transversal disruption of sarcomeres at N(2)-line level, longitudinal fissure of myofibrils, and loss of transversal alignments of Z- and M-lines) changes in myofibrils to rabbit (Oryctolagus cuniculus L.) meat tenderisation, during the ageing period (9 days at +4?°C), was studied for different types of muscle (type I, semimembranosus proprius; type IIB, semimembranosus accessorius; and type IID, psoas major). The results strongly suggest that myofibrillar structure weakening at N(2)-line level (evaluated by myofibrillar fragmentation upon mechanical homogenisation and observed by transversal disruption of sarcomeres), which is very likely mediated by cysteine endopeptidases, might be the major structural change responsible for rabbit meat tenderisation during ageing. Both myofibrillar fragmentation and transversal disruption of sarcomeres are good ageing indices for rabbit meat. The other major ultra-structural changes in myofibrils appear to have no major role in rabbit meat tenderisation at refrigeration temperatures. Finally, it is proposed that meat tenderisation during ageing depends mainly on the specific cleavage of titin molecules/filaments and nebulin molecules, at their susceptible sites located at or very close to the N(2)-line region (extensible segment and near C-terminus, respectively), mediated by cysteine endopeptidases (possibly calpains).     

31—THE EFFECT OF THE APPLICATION OF BATCHING OIL ON THE FINE STRUCTURE AND MECHANICAL PROPERTIES OF JUTE

An investigation is described in which jute filaments obtained from breaker-card sliver were subjected to the following treatments: (i) samples were made moisture-free but retained the batching oil; (ii) samples were made both moisture-free and oil-free; (iii) moisture-free and oil-free samples were soaked in water and again made moisture-free. Jute filaments were also obtained from raw fibre and from raw fibre from which natural fat and wax had been removed. The X-ray crystallinity was determined for all the samples in the moisture-free condition. The results obtained are explained on the basis of a paracrystalline structure of cellulosic fibre, some regions of which are capable of diffracting X-rays and at the same time are accessible to moisture. It has been proposed that, when the fibre is treated with an oil–water emulsion, water molecules penetrate into the amorphous regions and into portions of the region of intermediate order (paracrystalline), the resultant swelling then producing fissures that oil particles in the emulsion enter and in which they anchor themselves like wedges. These anchored oil particles are retained when the absorbed moisture (the water molecule) gradually evaporates to leave the structure in a less crystalline state. Rigidity values of the variously treated jute filaments support this view.     

不同钙离子浓度体外条件下磷酸化对肌联蛋白降解的影响

以羊背最长肌中肌联蛋白为原料,添加蛋白激酶A和碱性磷酸酶体外孵育使肌联蛋白发生磷酸化和去磷酸化反应。反应后添加μ-钙蛋白酶,在钙离子浓度分别为0.05 mmol/L和2 mmol/L条件下,4 ℃孵育2 d。测定孵育体系pH值、肌联蛋白磷酸化水平及其降解程度。结果表明：不同处理组孵育体系pH值差异不显著（P＞0.05）;蛋白激酶A组肌联蛋白磷酸化水平显著高于对照组,对照组肌联蛋白磷酸化水平显著高于碱性磷酸酶组（P＜0.05）;当钙离子浓度为0.05 mmol/L时,蛋白激酶A组和对照组未检测到分子质量为1 200 kDa的降解条带,而碱性磷酸酶组在孵育12 h检测到该条带;当钙离子浓度为2 mmol/L时,蛋白激酶A组和对照组均在孵育12 h检测到1 200 kDa降解条带,而碱性磷酸酶组在孵育0.5 h时检测到该条带。结论：蛋白激酶A和碱性磷酸酶能够促进肌联蛋白发生磷酸化和去磷酸化反应;随着钙离子浓度增加,肌联蛋白降解速率加快;并且在相同钙离子浓度下,肌联蛋白在碱性磷酸酶组降解较快,表明去磷酸化可以促进肌联蛋白的降解。     

Thermal properties of titin from porcine and bovine muscles

The thermal properties of titin isolated from porcine and bovine longissimus muscles were investigated by differential scanning calorimetry in the temperature range from 20 to 100?°C. A single peak with average maximum temperatures of 75.6 and 78.4?°C characterized porcine and bovine titin denaturation, respectively. The peaks were much broader than those from the other major muscle proteins. Titin denaturation enthalpy values (1.6-2.6 J/g) were only about half those of whole meat and also lower than those previously determined for myosin, actin, or collagen. The relatively high titin denaturation temperature suggests that it may be partially responsible for meat toughening when muscle tissue is heated above 60?°C.     

Molecular and structural biology of bleomycin and its resistance determinants

An anti-tumor antibiotic, bleomycin (Bm), causes cell death as a result of multiple strand scissions by direct interaction with bacterial and tumor cell DNAs. Some prokaryotic and eukaryotic cells have a system to protect themselves from Bm-induced toxicity. In eukaryotes, the response of normal and tumor cells to toxicity depends on the level of Bm hydrolase activity. The inactivation system of Bm, which hydrolyzes the amide in the beta-aminoalanine moiety of Bm, is also found in a few bacteria. We have shown that a Bm-resistance determinant, expressed in Bm-producing Streptomyces verticillus, the transposon Tn5 and methicillin-resistant Staphylococcus aureus, is a Bm-binding protein. A Bm N-acetylating enzyme, produced by S. verticillus, is also a Bm-resistance determinant. We have determined the X-ray crystal structures of Bm-binding proteins from S. verticillus and Tn5, designated BLMA and BLMT, respectively. Both crystal structures show that two Bm molecules bind to two Bm-binding pockets formed by the alternate arm exchange of two monomeric BLMA (BLMT) molecules. The Bm-binding proteins, complexed with Bm, are successfully crystallized and their X-ray crystal structures have been determined at high resolutions. The crystallographic analysis of the complexed protein gives a mode for binding to Bm: this is the first report regarding the X-ray crystal structure of the Bm molecule.     

Pressure-induced changes in the connectin/titin localization in the myofibrils revealed by immunoelectron microscopy

Changes in the connectin/titin localization in post-mortem and pressurized chicken muscles were investigated by immunoelectron microscopy. The anti-connectin monoclonal antibody, 1D11, strongly labeled the sides of thick filaments near the H-zone and weakly labeled the sides of Z-line in the sarcomere prepared immediately after death. With the development of the muscle contraction, the shortening of the sarcomere and the dispersion of the connectin epitope near the H-zone were observed. With the gradual increase of the sarcomere length during further storage, the apparent increase of the width of the epitope in the A-band region stained by the antibody was observed, but the distance from the epitope to M-line remained almost the same length. In the case of high pressure treatment, significant changes in the labeling pattern of the antibody were observed with the increase of the pressure applied. The increase of the distance from the epitope to M-line and dispersion of the epitope were observed in the fiber pressurized at 100 MPa. These phenomena were accelerated with the increase of the pressure applied. The discontinuous dense materials labeled by the antibody at the thick filament near the H-zone were observed in the fiber bundles pressurized at 200 MPa or more. This is probably due to the accumulation of connectin molecule from ordinary location in the sarcomere, because of the pressure-induced destruction of the thick and connectin filaments. In the fiber bundles pressurized at 300 MPa, a significant increase in the distance from the epitope to M-line accompanied with the increase of the sarcomere length was observed. From the results obtained, it was clear that the changes in the location of the connectin epitope induced by the brief exposure to high pressure were drastic in comparison with that in the sarcomere during post-mortem storage.     

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.     

On the mechanism of water holding in meat: The swelling and shrinking of myofibrils

Water holding in meat has, in the past, been rather poorly understood and has not been explained at all in structural terms. A unifying hypothesis for this phenomenon is that gains or losses of water in meat are due simply to swelling or shrinking of the myofibrils caused by expansion or shrinking of the filament lattice.

Myofibrils have been observed by phase contrast microscopy, and are seen to swell quickly to about twice their original volume in salt solutions resembling those used in meat processing. Such swelling is highly co-operative. Pyrophosphate reduces very substantially the sodium chloride concentration required for maximum swelling. In the absence of pyrophosphate, swelling is accompanied by extraction of the middle of the A-band; in its presence the A-band is completely extracted, beginning from its ends.

We suppose that Cl⁻ ions bind to the filaments and increase the electrostatic repulsive force between them. A crucial factor in swelling is likely to be the removal at a critical salt concentration of one or more transverse structural constraints in the myofibril (probably crossbridges, the M-line or the Z-line) allowing the filament lattice to expand.

We also point out that water losses in rigor, in the PSE condition and on cooking may well result directly from shrinkage of the filament lattice.     

Change in Titin Position in Postmortem Bovine Muscle

Myofibrils were prepared from bovine psoas muscles removed from the carcass at 3 and 48 hr postmortem and subsequently stained with a monoclonal antibody against titin. The antibody stained 2 bands per sarcomere (perpendicular to the fiber direction) in myofibrils from 3-hr muscle but often revealed 4 bands per sarcomere in the 48-hr samples. The results suggested that (1) the titin shape might be altered within the first 2 days postmortem, or (2) proteolysis of titin or a protein to which it was attached occurred.     

Titin content of beef in relation to tenderness

Steaks obtained from the longissimus dorsi muscle of 24 crossbred steers were subjected to four treatments (unaged raw, aged raw, unaged cooked, aged cooked) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Titin migrated primarily as a single protein band in unaged raw samples (48 h post mortem), as a doublet in aged (16 days) raw samples, and as a triplet in unaged and aged cooked samples. Total titin band density remained constant among steaks that varied widely in Warner-Bratzler shear value, suggesting that beef steaks varying in tenderness contain the same amount of titin. It is concluded that titin content, as determined by gel electrophoresis, does not distinguish 'tough' from 'tender' beef.     

Fuzzy neural network-based prediction of the motif for MHC class II binding peptides

Characterizing the interaction between major histocompatibility complex (MHC) molecules and antigenic peptides is critical for understanding immunity and developing immunotherapies for autoimmune diseases and cancer. To identify the peptide binding motif and predict peptides that bind to the human MHC classII molecule HLA-DR4(*0401), we applied a fuzzy neural network (FNN) capable of extracting the relationship between input and output. Analysis of the peptide binding motif revealed that the hydrophilicity of the position 1 residue located on the N-terminal side of the nonamer (9mer) was the most important variable and that the van der Waals volume and hydrophilicity of the position 6 residue and the hydrophilicity of the position 7 residue were also important variables. The estimation accuracy (A(ROC) value) was high and the binding motif extracted from the FNN agreed with that derived experimentally. This study demonstrates that FNN modeling allows candidate antigenic peptides to be selected without the need for further experiments.     

SDS-PAGE Conditions for Detection of Titin and Nebulin in Tender and Tough Bovine Muscles

Purified myofibrils were prepared from infraspinatus (tender) and rhomboideus (tough) muscles at 7 days postmortem and examined for myofibrillar/cytoskeleta1 protein degradation by using sodium dodecyl sulfate polyactylamide gel electrophoresis (SDS-PAGE). Four acrylamide/bisacrylamide ratios (37:1, 50:1, 75:l and 100:1) and two SDS-PAGE gel buffers (Tris-HCl, pH 8.0 and 8.9) were used to determine the optimum conditions for detection of titin and nebulin. Titin was degraded to a greater extent in myofibrils from the infraspinatus than in myofibrils from the rhomboideus. Very little nebulin was detected in either muscle. Use of acrylamide/bisacrylamide ratio of 37:1 and a gel buffer of pH 8.0 provided the most optimum conditions for detecting differences in the resolution of titin, nebulin and their apparent degradation products.     

Effect of Postmortem Storage on Titin and Nebulin in Pork and Poultry Light and Dark Muscles

Purified myofibrils were prepared from samples of chicken breast and thigh muscles and from light and dark portions of pork semitendinosus muscle at death and after storage at 4° and 22°C for 1, 3, and 7 days postmortem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the effect of postmortem storage of muscle on titin and nebulin. Results indicated that titin and nebulin were more rapidly degraded in light than in dark chicken muscle. In contrast, titin and nebulin were more rapidly degraded in dark than in light pork muscle.     

The effect of glycosylation on emulsifying and structural properties of bovine beta-casein

A number of attempts have been made to improve the functional properties of milk proteins by chemical modifications. One of such modifications is glycosylation which was carried out to determine the effect of covalent binding of glucose molecules to beta-casein (beta CN) on its emulsifying properties. It was found that up to six molecules of glucose were bound to one molecule of beta CN. Glycosylated beta CN produced smaller emulsion droplets than the intact beta CN. Increases in emulsion-forming and -stabilizing properties were observed. A prerequisite of proteins to form emulsions is their adsorption onto oil/water interface. Therefore the secondary structure of intact and glycosylated beta CN, both in solution and adsorbed onto a hydrophobic teflon/water interface also were studied by far-ultra violet circular dichroism (CD). It appeared, that after glycosylation the degree of helicity of intact beta CN decreased and the incorporation of glucose moieties most likely resulted in a type beta-turn formation after adsorption onto the interface.     

An immunological method to assess protein degradation in post-mortem muscle

A method for determining proteolysis of any specific protein in muscle is demonstrated. The protocol involves the preparation of a specific antibody which is used in immunoblotting total protein extracts from post-mortem muscle. In the present study an antibody to bovine myosin heavy chain was prepared that reacts with intact myosin heavy chain as well as proteolytic fragments that are generated by proteases. We show that little, if any, myosin heavy chain fragments are generated during prolonged aging of muscle at 4°C. In contrast, storage of muscle at 37°C results in the rapid breakdwon of myosin heavy chain to immunologically detectable peptides. Using a monoclonal antibody to titin, we demonstrate that this protein is degraded at 4°C during the aging period, and that, between 2 and 3 weeks following slaughter, no undergraded titin is detectable. This method is suitable for the analysis of any protein that can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE).     

Synthesis of β‐Galactooligosaccharides from Lactose Using Microbial β‐Galactosidases

Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β‐galactosidase, where the glycosyl group of one or more D‐galactosyl units is transferred onto the D‐galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β‐galactosidase enzyme or as agents to produce GOS molecules. Commercial β‐galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.     

7种氨基酸与丙烯醛典型反应产物的表征

为探究油炸薯片中的内源有害物丙烯醛产生后显著消减的原因,本研究利用马铃薯中7种含量较高的氨基酸(甘氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、缬氨酸、苏氨酸)与丙烯醛在不同温度及时间条件下反应,对反应产物进行分离纯化和结构鉴定,研究氨基酸对丙烯醛的清除作用及其作用机理.研究发现,所选氨基酸可高效消除丙烯醛,并生成8种首...