Postmortem proteome changes of porcine muscle related to tenderness

Proteome analysis was used to investigate the relation between changes in postmortem proteome of porcine muscle and tenderness development. Muscle samples were taken at slaughter and 72 h postmortem, and the registered changes in the proteome were related to Warner-Bratzler shear force. One hundred and three protein spots were found to change significantly (P < 0.01) over time, and of these the 27 most pronounced changes were identified. Eleven out of the 27 changes were fragments of actin. Other identified myofibril proteins or fragments included myosin heavy chain, titin, myosin light chain I, myosin light II, CapZ, and cofilin. Correlation analysis revealed significant correlations between three of the identified actin fragments and the myosin heavy chain fragment to shear force. Moreover, myosin light chain II and triose phosphate isomerase I were also found to correlate significantly to shear force. The results clearly demonstrate that postmortem degradation of actin and myosin heavy chain is related to meat tenderness.     

Separation and characterization of the constituents of compost and soil humic acids by two-dimensional electrophoresis

To investigate the chemical heterogeneity of humic acids (HAs), we applied two-dimensional (2-D) electrophoresis to HAs from a compost and two types of soils. In this method, HAs are first separated by isoelectric focusing (IEF) and then separated by polyacrylamide gel electrophoresis (PAGE). IEF and PAGE were carried out in the presence of 7?M urea. Upon 2-D electrophoresis of HAs, dark-colored substances were spread out across the gel mainly in the isoelectric point (pI) range of 3.0–4.5. Green fluorescence was observed in the smaller molecular size region of the gel, especially in the pI range of 3.0–4.5, and the most intense fluorescence was found at the moving front. The gels were divided into 36 sections, and then HA constituents were extracted from the individual sections and recovered by precipitation with acid. The distribution of organic carbon (C) among the gel sections coincided with that of the dark-colored substances on the gel. The total C recoveries were only 43–50%, suggesting that a considerable amount of HA constituents was lost during the extraction from the gels and purification. High-performance size-exclusion chromatography confirmed that the constituents of HAs were separated based on their molecular sizes by PAGE. The measurement of diffuse reflectance infrared Fourier transform (DRIFT) spectra indicated that the chemical properties of the HA constituents differed depending on the position on the gels and were affected by the molecular size rather than the pI. The fractions of the compost HA were characterized by higher proportions of aliphatic, proteinous and polysaccharide moieties and by the presence of lignin-derived structures. For the soil HAs, the fractions were characterized by a high proportion of the carboxyl group and a low proportion of aliphatic moieties. The proportion of proteinous and polysaccharide moieties in the fractions of soil HAs decreased with decreasing molecular size. The chemical properties of the green fluorescent substances remained unclear, since there was not enough of the substances to measure the DRIFT spectra. The present study showed that 2-D electrophoresis in the presence of concentrated urea offers an effective method for fractionating and isolating the constituents of HAs.     

Postmortem changes in pork muscle protein phosphorylation in relation to the RN genotype

Postmortem changes in pork muscle protein phosphorylation in relation to the RN(-) genotype were investigated using one-dimensional gel electrophoresis and a phosphor specific staining. The phosphorylation levels of several protein bands were found to be affected by the RN(-) genotype and to change during postmortem development. Glycogen phosphorylase, phosphofructokinase, and pyruvate kinase were found in protein bands affected by the RN(-) genotype, and the phosphorylation profile indicates that part of the increased rate and extended pH decline of the RN(-) genotype could be a consequence of phosphorylation of these key enzymes during the postmortem metabolism. The results illustrate that the protein phosphorylation level of the muscle proteins could be interpreted as a global metabolic fingerprint containing information about the activity status of the enzymes in the postmortem metabolism.     

Occurrence of Major Whey Proteins in the pH 4.6 Insoluble Protein Fraction from UHT-Treated Milk

A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.     

Competitive adsorption of water soluble plasma proteins from egg yolk at the oil/water interface

Water soluble plasma proteins were fractionated from hen's egg yolk, and the molecular weight and pI of the most abundant protein species were characterized with gel electrophoresis. The proteins were identified by mass spectrometry. The protein fraction was used to produce oil-in-water emulsions, both at various protein concentrations and at various pH values, and the surface load was determined through serum depletion. The competitive adsorption was studied through the determination of nonadsorbing species with gel electrophoresis. The results show that it was possible to form an oil-in-water emulsion for which droplet size and maximum surface load depended on the protein concentration and pH. Serum albumin and YGP40 adsorbed selectively at the oil/water interface throughout the pH range investigated, and for albumin the selectivity increased close to its pI. It is suggested that this selective adsorption is due to long hydrophobic stretches in the polypeptide chain, which are present in the selectively adsorbing species but absent in less adsorbing species.     

Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex)

It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.     

Preliminary study on puffer fish proteome-species identification of puffer fish by two-dimensional electrophoresis

The aims of this work were to determine the differential characterization of the urea soluble protein components of puffer fish species and to establish a preliminary proteomic database using an immobilized pH gradient two-dimensional electrophoresis (2DE) technique. The puffer fish muscle proteins resolved into 171-260 spots in the 2DE gels, with a pI range of 3-10 and molecular mass range of 7.4-205.0 kDa, following Comassie blue staining. Puffer fish muscle proteins fell in the region with pI values of 3.5-7.0, and molecular masses of 7.4-45.0 kDa were well-resolved and were good for species comparison. The more acidic proteins of lower molecular masses showed species specific characteristics. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic 2DE protein patterns.     

两蜂种蜂王浆蛋白质组分析

采用蛋白质组双向电泳技术和已鉴定蜂王浆蛋白质组的比对,对王浆高产蜜蜂（浆蜂）和中华蜜蜂（中蜂）的蜂王浆蛋白质组进行了差异比较。结果表明,浆蜂蜂王浆的总蛋白质点数（93个）显著高于中蜂蜂王浆总蛋白质点数（70个）,它们的等电点主要集中在5-8之间,分子量在50-80kDa之间,其中共有蛋白质点为30个;通过与已鉴定的浆蜂蜂王浆蛋白质组比较,这些共有蛋白质点推断为蜂王浆主蛋白1、2、3和葡萄糖氧化酶。同时两蜂种蜂王浆的蛋白质丰度也存在较大差异,共有点中有4个蛋白质的丰度中蜂显著高于浆蜂,7个浆蜂显著高于中蜂中。结果表明浆蜂和中蜂蜂王浆蛋白质种类及丰度均有较大差异。     

Gelation of chicken pectoralis major myosin and heat-denatured beta-lactoglobulin

Thermal, rheological, and microstructural properties of myosin (1 and 2% protein) were compared to mixtures of 1% myosin and 1% heat-denatured beta-lactoglobulin aggregates (myosin/HDLG) and 1% myosin and 1% native beta-lactoglobulin (myosin/beta-LG) in 0.6 M NaCl and 0.05 M sodium phosphate buffer, pH 6.0, 6.5, and 7.0 during heating to 71 degrees C. Thermal denaturation patterns of myosin and myosin/HDLG were similar except for the appearance of an endothermic peak at 54-56 degrees C in the mixed system. At pH 7.0, 2% myosin began to gel at 48 degrees C and had a storage modulus (G') of 500 Pa upon cooling. Myosin/HDLG (2% total protein) had a gel point of 48 degrees C and a G' of 650 Pa, whereas myosin/beta-LG had a gel point of 49 degrees C but the G' was lower (180 Pa). As the pH was decreased, the gel points of myosin and myosin/HDLG decreased and the G' after cooling increased. The HDLG was incorporated within the myosin gel network, whereas beta-LG remained soluble.     

空间诱变水稻多蘖矮突变体不同分蘖期蛋白质组研究

JB-1返回式卫星搭载水稻(Oryza sativa L.)丙95-503干种子,地面种植后筛选出多蘖矮杆突变体R955.利用荧光差异显示双向电泳(2-D DIGE)对突变体播种后第14天(未发生分蘖)、第21天(分蘖起始)、第55天(最高分蘖期)3个营养生长期叶片总蛋白进行分离及定量分析(未搭载植株为对照),检测到在...     

Denaturation and aggregation of myosin from two bovine muscle types

The thermal behaviors of myosin from bovine vastus intermedius (VI, predominantly red muscle) and semimembranosus (SM, predominantly white muscle) at pH 6.05 (ultimate pH of VI muscle) and 5.50 (ultimate pH of SM muscle) were compared. Differential scanning microcalorimetry and turbidity measurements were used to monitor changes in myosin during heating from 25 to 80 degrees C at 1 degrees C/min. VI and SM myosin heavy chain isoforms were identified on gradient SDS-PAGE. Endotherms of VI myosin at pH 6.05 had three transition temperatures (T(m)) of 45, 53, and 57 degrees C, whereas at pH 5.50 two transitions were observed at 42 and 59 degrees C. SM myosin had two T(m) values of 46 and 58 degrees C at pH 6.05 and T(m) values of 43 and 62 degrees C at pH 5.5. SM myosin at its ultimate pH was less heat stable than VI myosin at its ultimate pH; however, when SM and VI myosin were compared at the same pH, VI myosin was less stable.     

Comparison of different electrophoretic separations of hen egg white proteins

The hen egg white protein composition has not yet been fully defined. To improve the knowledge of this biological fluid, the most usual and recently developed electrophoretic methods have been used: SDS-PAGE, native-PAGE, isoelectric focusing (IEF), and 2-dimensional electrophoresis (2DE). Seven of the major known proteins were thus identified in at least one electrophoretic system. Isoforms of ovotransferrin, ovalbumin, and ovomucoid were visualized when pI was used for the separation. Two-dimensional electrophoresis allowed separation of a very large number of spots. In each of the four systems, some components were revealed but not identified, and unknown spots were particularly numerous with 2DE. With this technique, many spots corresponding to small acidic proteins were highlighted, among which was the Ch21 protein, whose presence in hen egg white was thus confirmed. This study thus constitutes, to our knowledge, the first proteomic investigation of hen egg white.     

Relationship between kinase phosphorylation, muscle fiber typing, and glycogen accumulation in longissimus muscle of beef cattle with high and low intramuscular fat

The objective of this study was to examine the association of adenosine monophosphate (AMP)-activated protein kinase (AMPK) with glycogen content in bovine muscle and their links with intramuscular fat (IMF) and muscle fiber type composition. Five steers with high intramuscular fat (High IMF, IMF content is 5.71 +/- 0.36%) and five steers with low intramuscular fat (Low IMF, IMF content is 2.09 +/- 0.19%) in the longissimus thoracis muscle (LM) were selected for immunoblotting, glycogen, and myofiber type composition analyses. The glycogen content was higher in Low IMF muscle than in High IMF muscle (1.07 +/- 0.07 versus 0.85 +/- 0.08 g/100 g muscle, P < 0.05). Phosphorylation of the AMPK alpha subunit at Thr 172, which is correlated with its activity, was lower (P < 0.05) in High IMF compared to Low IMF. In agreement with the lower AMPK phosphorylation in High IMF muscle, the phosphorylation of acetyl-CoA carboxylase (ACC) was also lower (P < 0.05) in High IMF muscle than in Low IMF muscle. Glycogen synthase kinase 3 (GSK3) down-regulates glycogen synthesis through phosphorylation of glycogen synthase. The phosphorylation of GSK3 in High IMF was lower (P < 0.05) than that in Low IMF, which should down-regulate glycogen synthase activity and reduce the glycogen content in High IMF beef. Type IIB myosin isoform was absent in beef muscle. No noticeable difference in myosin isoform composition was observed between Low and High IMF muscle. In summary, High IMF cattle had lower LM glycogen levels than low IMF cattle, and AMPK activity was less in High IMF than in Low IMF cattle. The difference in glycogen content between Low and High IMF muscle was not correlated with muscle fiber composition. This data shows that LM lipid and glycogen metabolisms are affected by AMPK activity. Thus, AMPK may be a molecular target to alter IMF and glycogen levels in beef muscle.     

Monitoring of denaturation processes in aged beef loin by Fourier transform infrared microspectroscopy

We present the results of a Fourier transform infrared (FT-IR) microspectroscopic study using conventional FT-IR microscopy and FT-IR imaging to detect the denaturation process during four different heating temperatures (raw, 45, 60, and 70 degrees C) spatially resolved in bovine cryosections from longissimus dorsi muscle. FT-IR imaging, employing a focal plane array detector, which allowed the simultaneous collection of spectra at 4096 pixels, enabled the investigation of the heat-induced changes in the two major meat constituents, i.e., myofibrillar and connective tissue proteins, spatially resolved. The infrared spectra of both compounds revealed that the major spectral changes involved an increase in beta-sheet and a decrease in alpha-helical structures, which appeared to be much more pronounced for the myofibers than for the connective tissue. These conformational changes could be correlated to the denaturation of the major meat proteins, such as myosin, actin, and collagen.     

杀雄剂SQ-1诱导小麦雄性不育花药蛋白质组分分析

采用固相pH梯度SDS聚丙烯酰胺双向凝胶电泳技术对杀雄剂SQ-1处理和未处理的小麦单核期、二核期花药总蛋白进行了分离，通过考马斯亮蓝G-250染色，获得了分辨率和重复性较好的双向电泳图谱。通过PDQuest 2DE图像分析软件的分析，在等电点(pI)4～7之间可识别约500个以上较为清晰的蛋白质点，处理和对照各时期蛋白质在等电点5～6，分子量20～40kD之间相对较集中；检测到单核期差异点43个，二核期差异点45个，并计算出了各差异点的分子量和等电点。其中11个点在处理材料的单核期缺失而在对照中表达，7个在处理中表达而在对照中缺失，14个点表达量在处理中明显减弱，而另外11个明显增强；在二核期45个差异点中，有13个在处理中缺失而在对照中表达，7个在处理中表达而在对照中不表达，12个表达量在处理中明显减弱而另外13个明显增强。经SQ-1处理后在单核期和二核期A-Z3（26.8/5.7）和C-Z5（26.8/5.7）点均缺失。点A-L10(26.8/5.9)在单核期处理的表达量下调，而到了二核期完全缺失C-Z6（26.9/5.9）点。通过双向电泳技术获得的这些差异蛋白可能直接或间接地参与了花药发育的各个途径，因此，处理和对照图谱中表现出的差异蛋白很可能与SQ-1诱导小麦雄性不育有关。     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率（浓度）高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.     

Detection of giant myofibrillar proteins connectin and nebulin in fish meat by electrophoresis in 3-5 gradient sodium dodecyl sulfate polyacrylamide slab gels

An improved method was investigated for sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) to facilitate the analysis of the giant myofibrillar proteins, connectin and nebulin, in fish meat by using jack mackerel (Trachurus japonicus) as the sample fish. It was established that separation of the alpha-connectin band from the beta-connectin band by SDS-PAGE could be achieved by using 3-5% gradient gels with glycerol to facilitate the formation of a gradient with polymerization at 35 degrees C. SDS-PAGE samples of white dorsal muscle from the jack mackerel were homogenized with a 2% SDS solution containing an inhibitor mixture (1 microg/mL of phenylmethanesulfonyl fluoride, 1 microg/mL of leupeptin, and 1 microg/mL of E-64) and heated at 50 degrees C for 20 min. Heating these samples at 100 degrees C for 2 min resulted in the disintegration of connectin but did not affect nebulin. A purified myofibril sample and a whole muscle sample showed similar changes in the overall rate of degradation of whole connectin and nebulin during the postmortem storage period, but it was clear that beta-connectin was cleaved from alpha-connectin during the preparation of myofibrils at the early stage postmortem. Storage of the SDS-PAGE samples at -85 degrees C was preferable to storage at -18 degrees C for a long period.     

Effects of proteolysis and mechanism of gel weakening in heat-induced gelation of fish myosin

Addition of papain decreased the onset temperature and the rate at which G' developed during heat-induced gelation of arrowtooth flounder myosin. Frequency sweep results revealed that G' markedly decreased in proportion to the amount of papain added. However, use of E-64, a cysteine proteinase inhibitor, reversed the effects of papain and protected myosin heavy chain from degradation. DSC thermograms indicated papain significantly decreased the enthalpy required to induce myosin denaturation without significant changes in onset and maximum transition temperatures. Thermal denaturation kinetics indicated decreases in both the activation energy and the rate of myosin denaturation. CD studies revealed a rapid decrease in alpha-helical content, indicating the initial degradation of myosin molecules mostly occurred in the tail region. These results suggested that proteolysis affected thermal properties and reactivity of myosin during heating. Although myosin gel could be formed, structural disruption resulted in lower gelling ability and rigidity of the formed gel.     

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.