Study of DNA-Binding Proteins of Rat Liver Mitochondria

Acid-soluble proteins able to form DNA-protein complexes in the presence of physiological concentration of NaCl were isolated from rat liver mitochondria. Electrophoretic analysis of these proteins in 15% polyacrylamide gel showed that mitochondrial acid-soluble proteins include of approximately 20 polypeptides with molecular weight of 10–120 kDa. The fraction of acid-soluble proteins can be separated into basic and acidic proteins by chromatography on DEAE cellulose. Some of acidic proteins are tightly bound to the basic proteins and can be separated from them in the presence of 5 mM dithiothreitol. It is discovered that the fraction of acidic proteins contains proteases (including DNA-activated ones), which cleave different polypeptides of the basic proteins with different efficiency. Possibly, mitochondrial DNA-binding proteins and DNA-activated proteases are involved in the regulation of structural organization and functional activity of mitochondrial DNA.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

Distinguishing Small Molecular Mass Differences of Proteins by Mass Spectrometry

Electrospray ionization–Fourier transform ion cyclotron resonance (ESI–FTICR) mass spectrometryallows for high-resolution, accurate mass analysisof multiply charged ions of proteins. In the workdescribed here, the ability of ESI–FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochromec, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based onthe tallest isotopic peak of each ubiquitin chargestate. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and thetallest peak was easily identified. The peak massesof cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observedfor some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge statesdid not yield an unequivocal assignment of thetallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.     

Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.     

大鼠肝线粒体中的脂肪酸分析

用双 2 乙基己基酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,然后用蔗糖密度梯度离心法分离大鼠肝线粒体 ,用毛细管气相色谱法测定肝线粒体中的脂肪酸含量。测定结果 :所测 1 4种脂肪酸的总量 ,青年正常组大于青年诱导组 (P <0 .0 1 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。不饱和脂肪酸与脂肪酸总量的比例 ,老年诱导组大于老年正常组 (P <0 .0 5 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。长链脂肪酸与脂肪酸总量的比例 ,老年正常组小于老年诱导组 (P <0 .0 5 )。结果表明 ,用DEHP诱导大鼠肝过氧化物酶体增值 ,影响肝线粒体脂肪酸正常代谢 ,使线粒体膜结构发生变化 ,这种变化 ,青年鼠与老年鼠不同     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

Mitochondria Isolated from Rat Brown Adipose Tissue and Liver

Mitochondrial fractions, relatively free from contamination by other cytoplasmic structures, have been isolated by differential centrifugation from homogenates of brown adipose tissue from starved rats. It was possible in such fractions to distinguish two types of mitochondria in this tissue. Type I mitochondria, when morphologically intact, are limited by a bilaminar membrane and show regular parallel cristae. In isolated fractions, a proportion of these mitochondria are swollen, vacuolation occurring within the cristae between their limiting membranes. Type II mitochondria are distinguished from the more numerous type I bodies by the opaque appearance of their matrix. They are limited by a membrane which is in part single, and in part double. They show a few, but crisply outlined internal membranes. Vacuolation of this type of mitochondrion has not been observed. Vacuolation comparable to that in brown fat mitochondria was also observed between the two laminae of the enclosing membrane and within the cristae of liver mitochondria.     

Isolation of Rat Liver Mitochondria and of Their Ribonucleic Acid

Mitochondria prepared from normal or regenerating rat liver appeared homogeneous on examination by electron microscopy. Ribonucleic acid (RNA) isolated from such mitochondria by phenol extraction or by deproteinization without phenol was resolved on sucrose density gradients into 18S, 12S and 4S optical density peaks. Administration of 5-[³H]-uridine to normal or partially hepatectomixed animals for 16 hours resulted in the labeling of A IS, 36S, 28–29S, 14S, 9–10S and 4S RNA species. Labeling of 18S RNA from regenerating liver but not from normal liver was also observed.     

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mm ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin β₄, and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.There is an urgent need for tools to comprehensively identify markers of normal and pathological processes at the molecular level. DNA microarrays have enabled researchers to follow gene expression changes with respect to many of these processes, including individual tumors in the case of cancer (1). Direct detection of proteins is typically required to validate changes at the gene product level; however, the changes in protein levels do not always reflect changes in gene expression because of post-translational modifications, differential compartmentalization, recycling, and degradation. Because it is ultimately the proteins that convey cellular phenotypes, it is necessary to develop methods for direct screening of proteins, and mass spectrometry shows promise for this purpose. However, the usefulness of mass spectrometry as an analytical tool to detect proteins in cells or tissue is limited to the extent to which the sample is sufficiently enriched for the specific fraction of interest. It is still challenging to identify molecules involved in specific normal or pathological processes because the relevant proteins are often difficult to isolate from the majority of cellular proteins that are not correlated to the process of interest. In this context, an ideal proteomics approach would require a minimal amount of starting material, be amenable to an efficient enrichment strategy, and would provide results quickly.It has been well established that molecules directly involved in cell-cell and cell-substrate adhesions are critical for processes such as epithelial to mesenchymal transition and wound healing. Their further role in regulation of tissue integrity, cell polarity, motility, and invasion is emphasized by a variety of disorders stemming from their inappropriate expression and mutations (2, 3). Selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 have been established both as biomarkers (4) and predictive factors (5, 6) for the development of accelerated atherosclerosis and heart disease. In epithelial tissues, reduced expression of the cell-cell adhesion molecule E-cadherin correlates with epithelial to mesenchymal transition, tissue invasion, and metastasis and is a prognostic biomarker of poor clinical outcome in many cell types (7–9). Furthermore, up-regulating E-cadherin is considered as a treatment option in several types of cancer (10). Therefore, methods are also needed to not only identify adhesion molecules as disease markers but to also understand the pathology of underlying medical problems caused by impairment in adhesion molecule function (e.g. inability to heal chronic wounds (11)). However, the lack of knowledge about regulation and functional interactions of the specific adhesion-related proteins has so far thwarted the attempts at direct targeting of these molecules in basic and clinical research (12, 13). Therefore, a comprehensive understanding of how proteins that function in adhesive processes work together to maintain proper tissue form and function is critical.Some of the same barriers to effective application of mass spectrometry as an analytical tool (as discussed above) have impeded analysis of cell-cell and cell-matrix adhesion-dependent processes such as wound healing and cancer (14). The study of extracellular matrix (ECM)1 and adhesion-related proteins is further complicated by the difficulty in sample preparation because compared with cytosolic proteins basal cell proteins are often highly insoluble (e.g. transmembrane and plaque components) and difficult to isolate from intracellular proteins. One general strategy involves using ECM-specific enzymes to dislodge the cells at their points of attachment (15). The supernatant from the partial digest is collected for further proteomics analysis. However, most mass spectrometric analyses depend on detection of peptides with specific ionization and fragmentation properties that are most readily achieved using trypsin as the sole enzyme. The use of ECM-specific enzymes may result in a distribution of peptides that are not optimal for detection (i.e. the generation of non-tryptic termini). The other general approach to isolate components of the ECM involves using detergents to lyse cells on the surfaces to which they are attached and collect the remaining cell debris for analysis (15). Although progress has been made with respect to the creation of “mass spectrometry-friendly” detergents (16), the use of chemicals for the purpose of protein solubilization is generally not ideal. To overcome these problems, we adapted a fast, simple method of isolating extracellular, transmembrane, and associated proteins (from here on collectively referred to as “basal cell proteins”) from cells attached to a solid substrate. The method consists of “deroofing” the cells attached to glass coverslips by 20 mm NH₄OH solution followed by rapid water rinses to remove the bulk of the cell and its remaining debris (17). Our results show efficient removal of cytoplasm and organelles and detection of basal cell proteins by mass spectrometry, including those involved in cell-cell and cell-extracellular matrix interactions. These proteins were liberated from the surface with trypsin, and the subsequently generated peptides were detected and profiled for differences using LC-FTMS.The approach was first validated by comparing basal cell protein composition in mouse keratinocytes with or without a critical cell-cell junction protein called plakoglobin (PG). This desmosomal protein is required for cell-cell adhesion and maintenance of tissue integrity (18). Plakoglobin inhibits keratinocyte motility (19) and is down-regulated in several distinct tumor types, including bladder, breast, and cervical cancers (20–22). Moreover, we were able to dissect the molecular differences between an independent isolate of PG^−/− keratinocytes that behaved differently in motility assays from the rest of the PG-null cells, further emphasizing the potential for using the method to differentiate between cells with distinct adhesive and motile behaviors. The method was then evaluated in clinically relevant human tumor cell lines by extending the analysis to include two human oral squamous cancers of different origin. Because they lack precisely defined changes in cell adhesion molecules and phenotype, we compared the basal cell protein expression of UM-SCC-1 (23) and CAL33 (24) cell lines isolated from the roof of the mouth and tongue, respectively. These experiments revealed 40 proteins differentially expressed between the cell lines among over 100 detected. Moreover, the proteomic profile reveals a set of motility- and invasion-related genes unique to tongue-derived CAL33 cells. This could indicate the difference between oral cancers derived from different parts of the mouth, or it may indicate a potential difference in aggressiveness between these cell lines. These results show that our detection method is applicable for both detection and comparative studies in human cancer model systems.     

Absence of Nitric-oxide Synthase in Sequentially Purified Rat Liver Mitochondria

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[¹⁴C]arginine to l-[¹⁴C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.Nitric oxide (NO^·)² is a highly diffusible, hydrophobic, and gaseous free radical (1) that is responsible for autocrine and paracrine signaling activities (2). NO^· can readily partition into and through membranes (3–5) to influence biological functions such as blood pressure regulation, platelet aggregation and adhesion, neurotransmission, and cellular defense (4, 6–11). The mechanism by which NO^· influences biological functions is by binding to target proteins that contain heme and/or thiol(s). Alternatively, NO^· can combine with to produce the highly reactive species peroxynitrite.Mitochondria are highly compartmentalized, membranous organelles that contain abundant amounts of reactive hemoproteins and thiols (12, 13), to which NO^· may bind reversibly (14, 15) or irreversibly (16–18). Mitochondria also generate various amounts of during the process of cellular respiration (19, 20). Studies conducted during the past decade have suggested that NO^· can diffuse into mitochondria and cause mitochondrial dysfunction by reversibly inhibiting cytochrome c oxidase (14, 21, 22) and NADH dehydrogenase (23).In the mid-90s, a putative variant of NOS was proposed to reside within mitochondria. Initially, Kobzik et al. (24) and Hellsten and co-workers (25) observed an apparent endothelial NOS (eNOS) immunoreactivity in skeletal muscle mitochondria. Simultaneously, Bates et al. (26, 27) observed an apparent eNOS histochemical reactivity in inner mitochondrial membrane preparations, isolated from rat liver, brain, heart, skeletal muscle, and kidney. Tatoyan and Giulivi (28), acting on these initial observations, performed experiments in an attempt to confirm the identity of this putative mtNOS. Relying on immunochemical analysis, Tatoyan and Giulivi (28) claimed that inducible NOS (iNOS) was the NOS isoform present in rat liver mitochondria. This same group using mass spectrometry later presented data in support of the putative mtNOS being a variant of nNOS (29). Ghafourifar and Richter (30) had reported previously that the putative mtNOS was calcium-sensitive and constitutive in nature. Since these reports, different groups have reported the presence of each of the three main isoforms of NOS within mitochondria (29, 31, 32). Also, biochemical characterization of the putative mtNOS performed by Giulivi and co-workers (29) revealed certain post-translational modifications (myristoylation and phosphorylation of the protein) that are thought to be unique to eNOS. During the last decade, various reports have supported the presence of at least one of the three main isoforms of NOS residing in mitochondria. However, the more recent reports tend to question this claim (33–36). Because of the contradictory reports regarding the existence of a putative mtNOS, Brookes (33) compiled a critical and thorough review of the literature published up to 2003 dealing with the putative mtNOS. This review brought to light the diverse technical issues involved in the aforementioned studies. Major issues were the degree of purity of mitochondrial preparations (37, 38), shortcomings of measurement methodology (29, 39–41), use of inappropriate, or total lack of, experimental controls and confusing technical practices. Lacza et al. (42) has reviewed the more recent developments in the area of mitochondrial NO^· production and discussed some of the shortcomings of certain techniques still being used.In light of this ongoing controversy regarding the presence or absence of a mtNOS, we designed and carefully conducted properly controlled studies to either confirm or refute the existence of any NOS isoform within mitochondria. Ultrapure rat liver mitochondria were isolated using repeated differential centrifugation followed by Percoll gradient purification. Proteomic analyses were then performed using a nano-HPLC-coupled nanospray LTQ-MS. To avoid the interfering factors that are rampant in NO^· trapping assays (43), the NOS-catalyzed conversion of l-[¹⁴C]arginine to l-[¹⁴C]citrulline was used to probe for NOS activity in mitochondria. Appropriate controls were employed and, for inhibition studies, high concentrations of l-thiocitrulline (TC) (44) were used. Additionally, immunochemical analyses were performed with ultrapure mitochondria using nNOS, eNOS, and CaM antibodies. The problems faced with the commonly used techniques in mtNOS studies are discussed.     

nHAP对大鼠肝线粒体的生物活性作用

为了探讨羟基磷灰石纳米粒子(nHAP)对大鼠肝线粒体生物活性的影响,将nHAP直接作用于线粒体,在不同浓度和时间下测定线粒体标志酶琥珀酸脱氢酶(SDH)比活性,并与对照组进行比较。结果显示,当nHAP中水含量在10%以下时,线粒体生物活性未发现改变;当nHAP浓度递增时,在等时间段内,对线粒体SDH比活性呈逐步抑制作用;在不等时间段内,nHAP对线粒体SDH比活性的抑制作用与对照组相比较差异有显著性(p<0.05)。因此,nHAP对线粒体SDH比活性的抑制有浓度和时间的依赖性。     

小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

Detection of Protein Kinase A and C Target Proteins in Rat Brain Mitochondria

Phosphorylation of some membrane-bound proteins in the mitochondria of rat liver and brain is regulated by Ca²⁺ and cAMP acting as secondary messengers. These proteins are the main myelin components: 46 kDa 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) and two isoforms of the myelin basic protein (MBP) with molecular weights of 17 and 21.5 kDa, which we have identified previously and found outside myelin in rat brain mitochondria. The phosphorylation level of CNP and both MBP isoforms increases when the mitochondrial permeability transition pore (mPTP) is opened. It is known that protein kinases A and C in heart mitochondria are directly bound to mPTP regulator proteins and are able to modulate the pore function. It is shown in this study that the inhibitors of protein kinases A (H-89) and C (staurosporin, Go 6976, and GF 109203 X) decrease the phosphorylation level of CNP and two MBP isoforms allowing us to assume that they are the targets of the signaling protein kinases A and C.     

Streptolydigin, an Inhibitor of Oxidative Phosphorylation in Rat Liver Mitochondria

Streptolydigin interferes with oxidative phosphorylation in rat liver mitochondria. The agent acts primarily as an uncoupler of respiration-associated phosphorylation but also impairs respiration to various degrees depending on the substrate. Streptolydigin partially inhibits electron flow at a point past the cytochrome b and prior to the cytochrome c reduction site. Streptolydigin also inhibits the function of the enzyme ribonucleic acid polymerase in whole bacterial cells and cell-free systems. The streptolydigin concentrations that cause effective inhibition of ribonucleic acid polymerase in cell-free systems are approximately 10 times less than those required to inhibit oxidative phosphorylation in mitochondria.     

A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

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Highlights

• •XL-MS reveals new PPIs in yeast mitochondria under glycerol and glucose condition.
• •Significant but limited results from quantitative XL-MS experiments.
• •Ndi1 participates in a CIII₂CIV₂ respiratory supercomplex.
• •Min8 promotes assembly of Cox12 into an intermediate complex IV.

     

Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins

Electron capture dissociation (ECD) represents one of the most recent and significant advancements in tandem mass spectrometry (MS/MS) for the identification and characterization of polypeptides. In comparison with the conventional fragmentation techniques, such as collisionally activated dissociation (CAD), ECD provides more extensive sequence fragments, while allowing the labile modifications to remain intact during backbone fragmentation—an important attribute for characterizing post-translational modifications. Herein, we present a brief overview of the ECD technique as well as selected applications in characterization of peptides and proteins. Case studies including characterization and localization of amino acid glycosylation, methionine oxidation, acylation, and “top–down” protein mass spectrometry using ECD will be presented. A recent technique, coined as electron transfer dissociation (ETD), will be also discussed briefly.     

利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

大鼠急性应激时肝线粒体质子跨膜转运活性的调控

大鼠烫伤早期(烫伤后30min),肝线粒体质子和电子传递速度均加快,线粒体能化态跨膜电位降低(均以琥珀酸为底物),线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。