巴豆提取物诱导小鼠小肠组织中蛋白质差异表达的初步研究

目的 建立和优化蛋白质分子差异展示的双向聚丙烯酰胺凝胶电技术,初步观察巴豆提取物诱导小鼠小肠组织中蛋白质的差异表达,为进一步筛选其中介导巴豆生物作用的蛋白质分子奠定基础。方法 应用巴豆提取物灌胃制备小刀慢有肠运动增强模型,提取其小肠组织中总蛋白质,优化蛋白质分子差异展示的双向聚丙安凝胶电泳技术,并用其分析模型动物小肠组织中蛋白质,双向电泳凝胸银染法显示其蛋白质的差异表达。结果 建立了小鼠慢性胃肠运     

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20–30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.Laemmli’s system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences (1). The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in “overnight” adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered “the single most useful analytical tool to study protein molecules” (3), SDS/PAGE is routinely used for simultaneous determination of protein heterogeneity and molecular weight in applications ranging from diagnosis of hereditary red cell membrane disorders to evaluation of recombinant protein expression and purification procedures. Protein analysis by SDS/PAGE is relatively simple, affordable, and rapid (4): A buffer containing a tracking dye and SDS is added to the sample of interest, the mixture is applied to a polyacrylamide gel, and a potential difference is used to drive the dye and the resulting anionic particle composed of protein and dodecyl sulfate (DS) through the gel. The distance traveled by the protein/DS particle from the top of the gel is then divided by that of the dye to obtain relative migration (R_f), and molecular weight [as relative molecular mass (M_r)] determined by comparison of this value with a logarithmic plot derived from the R_fs and M_rs of reference proteins.Fractionation on SDS/PAGE is controlled by the molecular size and shape of the protein/DS particle, its net charge, and the accessible spaces among the acrylamide fibers that comprise the gel matrix as determined by the total concentration of acrylamide and bis-acrylamide cross-linker [T; Materials and Methods (5)]. Larger particles become trapped within the gel meshwork and migrate slower than smaller species. Low-percentage gels are therefore typically used to resolve larger proteins, and vice versa. Acrylamide concentrations compatible with routine use are usually from 4–20% T due to practical considerations, because gels outside of this range are too fragile or too brittle, respectively, to withstand the physical manipulation(s) required for protein visualization and/or immunoblotting.Most globular, water-soluble proteins are reliably identified by their SDS/PAGE mobility relative to corresponding reference proteins typically used to estimate molecular weight. However, this group of well-behaved polypeptides does not include helical transmembrane (TM) proteins, macromolecules that comprise 20–30% of the human genome (6), comprise the majority of drug targets (7), and are the focus of major pharmaceutical discovery efforts (8). For example, the first true G protein-coupled receptor to be determined to high resolution, 39-kDa bovine rhodopsin (9), migrates on SDS/PAGE to positions consistent with sizes as low as 30 kDa (10). In fact, we have previously shown that the gel mobility of helical TM proteins seldom corresponds to formula molecular weight (11). This phenomenon of “anomalous migration” can arise as a consequence of the high hydrophobicity and concomitant binding of DS by TM proteins at levels that exceed those of water-soluble polypeptides (12). However, quantitation of DS binding stoichiometry is not routine and consumes milligram amounts of purified samples. Thus, the impact of enhanced DS binding on the direction and magnitude of anomalous migration has remained unpredictable for decades, with helical TM proteins variously exhibiting gel mobility reduced, equivalent, or increased relative to reference proteins (11–13). Such differences are generally disregarded when protein identity is known or can be confirmed in orthogonal molecular weight determination procedures but, in many instances, raise questions of protein folding, oligomeric organization, proteolytic processing, posttranslational modification(s), alternative splicing, antibody cross-reaction, and/or degradation. These issues become acute in SDS/PAGE analyses of tissue or cell extracts, where reasonable molecular weight estimates remain crucial for protein identification.Here, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of de novo designed TM-mimetic peptide polymers with typical water-soluble reference proteins on Laemmli gels ranging from 11–18% T. We find that net charge and effective molecular size among the migrating TM-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms derived from these data compensate for the differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a straightforward means to predict anomalous migration of membrane proteins relative to reference polypeptides, facilitating their identification by molecular weight in SDS/PAGE applications.     

Blood Group-Active Surface Molecules of the Human Red Blood Cell

The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately 10(6) copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) had AB(H) activity and there are about 5 x 10(5) copies/red cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x 10(5) copies/red cell) are polypeptides of Mr 30,000, polypeptides of Mr 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of Mr 30,000 and those of Mr 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group 'N' antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately 10(5) copies/red cell. The complete protein sequence of all the above-mentioned proteins is known, except for the Mr 30,000 and Mr 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C. Several of the minor blood group active proteins at the red cell surface (abundance less than 1.2 x 10(4)/red cell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inb antigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.     

A strategy for the identification of proteins targeted by thioredoxin

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5-three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.     

Hybrid molecules containing the B-domain of insulin-like growth factor I are recognized by carrier proteins of the growth factor.

The insulin-like growth factors (IGFs) are polypeptides in plasma that are chemically related to insulin and have mitogenic and insulin-like activity. Unlike insulin, the IGFs circulate in plasma bound to specific high molecular weight carrier proteins that regulate their delivery to target tissues. To define the sites on the IGFs that allow them to be recognized by carrier proteins, we constructed hybrid molecules containing different portions of the insulin, IGF-I, and IGF-II molecules. The presence of the B domain of IGF-I, but not the D domain of IGF-II, enables these insulin-IGF hybrid molecules to be recognized by acid-stripped IGF carrier proteins from rat serum and other sources. By contrast, neither the BIGF-I nor DIGF-II domain is sufficient to enable binding to type II IGF receptors, despite the fact that type II receptors, like the carrier protein, specifically bind IGF-I and IGF-II but do not interact with insulin. By differentiating those sites on the IGF molecule required for binding to IGF carrier protein and receptors, the insulin-IGF hybrid molecules should help delineate the role of the carrier protein in presenting biologically active IGF to target tissues.     

Attempts to restore scrapie prion infectivity after exposure to protein denaturants.

A wealth of experimental evidence argues that infectious prions are composed largely, if not entirely, of the scrapie isoform of the prion protein. We attempted to restore scrapie infectivity after exposure to protein denaturants including urea, chaotropic salts, and SDS. None of the procedures restored infectivity. Dialysis to remove slowly chaotropic ions and urea failed to restore scrapie infectivity. Attempts to create monomers of the scrapie isoform of the prion protein under nondenaturing conditions using a wide variety of detergents have been unsuccessful, to date, except for one report claiming that scrapie infectivity could be recovered from 12% polyacrylamide gels after SDS/PAGE [Brown, P., Liberski, P. P., Wolff, A. & Gajdusek, D. C. (1990) Proc. Natl. Acad. Sci. USA 87, 7240-7244]. We found that < 0.001% of the infectious prion titer could be recovered from the region of a polyacrylamide gel where the denatured proteinase K-resistant core of the scrapie isoform of the prion protein and other 30-kDa proteins migrate. We conclude that under the denaturing conditions used for SDS/PAGE, the scrapie isoform of the prion protein is denatured and little or no renaturation occurs upon injection of fractions eluted from gels into animals for bioassays.     

Heart failure and protein quality control

The heart is constantly under mechanical, metabolic, and thermal stress, even at baseline physiological conditions, and cardiac stress may increase as a result of environmental or intrinsic pathological insults. Cardiomyocytes are continuously challenged to efficiently and properly fold nascent polypeptides, traffic them to their appropriate cellular locations, and keep them from denaturing in the face of normal and pathological stimuli. Because deployment of misfolded or unfolded proteins can be disastrous, cells, in general, and cardiomyocytes, in particular, have developed a multilayered protein quality control system for maintaining proper protein conformation and for reorganizing and removing misfolded or aggregated polypeptides. Here, we examine recent data pointing to the importance of protein quality control in cardiac homeostasis and disease.     

Identification and characterization of an abundant ovarian interstitial gland protein associated with sexual maturity in rabbits

An abundant ovarian protein with a relative mol wt (Mr) of 37K and an apparent pI of 8, associated with the onset of sexual maturity in the rabbit, has been identified. Ovaries from sexually mature (greater than 6 months old) rabbits contain large quantities of this 37K protein, while none can be detected in ovaries of immature (1 and 2 months) animals. Analysis of polyacrylamide gel electrophoresis (PAGE) gels of mature ovarian homogenates demonstrates that this protein is more abundant than actin in these preparations. It appears to be tissue specific, since it was not detected in 16 other rabbit tissues tested. Autoradiographic analysis of proteins labeled with 35S in ovarian organ culture demonstrates that a protein of identical Mr and charge to the 37K protein is synthesized in this tissue. Polyclonal sheep antiserum has been produced to the two-dimensional PAGE-purified protein. Immunoblotting of two-dimensional PAGE gels shows specific recognition of this protein and two slightly more acidic proteins of the same Mr by this antiserum. These three protein species also stain identical colors with a silver-based color stain, further suggesting that these are charge variants of the same protein. This protein is not present in corpora lutea isolated form sexually mature ovaries and is present in interstitial cell-enriched ovaries of rabbits which have been actively immunized with zona pellucida proteins. Immunocytochemical localization studies further demonstrate that this protein is localized in the interstitial gland cells. These findings suggest that this 37K protein is not associated with either follicular or luteal cells, but, rather, is linked with the 20 alpha-hydroxyprogesterone-secreting interstitial gland cell population.     

Antenatal diagnosis of β-thalassemia by isoelectric focusing in immobilized pH gradients

A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7–7.6 span, HbA and HbF_ac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6–8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of β-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -F_ac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous β-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.     

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.     

Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35S]methionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Two-dimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.     

Identification of the uvrC gene product.

We have constructed a multicopy plasmid that carries the uvrC gene of Escherichia coli. By inserting the transposon Tn1000 (previously designated gamma delta) into this plasmid, we obtained many derivatives that fail to complement uvrC34. The proteins synthesized by the original plasmid and the uvrC::Tn1000 derivatives were labeled in maxicells and analyzed on gels, demonstrating that a protein of Mr 70,000 encoded by the original uvrC+ plasmid was absent from the mutated noncomplementing derivatives; this protein is presumed to be the uvrC gene product. We found that this protein of Mr 70,000 binds to DNA and have partially purified the uvrC protein by DNA-cellulose chromatography. Because some of the uvrC::Tn1000 derivatives produce truncated polypeptides, the orientation of expression and the location of the promoter were determined by correlating the sizes of the truncated polypeptides with the sites of insertion of Tn1000.     

Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been prepared; it does not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate [Lane, D.P. & Robbins, A.K. (1978) Virology 87, 182-193] is directed primarily against determinants that are not sensitive to periodate.     

Electrophoretic purification of radioiodinated follicle-stimulating hormone for radioligand receptor assay and radioimmunoassay

A method is described for electrophoretic purification of [125I]human (h) FSH after radioiodination that improves radioligand binding to FSH membrane receptors. Lactoperoxidase-iodinated hFSH was separated from reaction products by electrophoresis on 7.5% polyacrylamide tube gels (PAGE). Material eluted from 3-mm gel slices was analyzed for incorporation of 125I and binding to antibody (RIA) or receptor (RRA), and by sodium dodecyl sulfate-PAGE for protein composition. Sodium dodecyl sulfate-PAGE analysis of individual PAGE fractions demonstrated that iodinated proteins, both higher and lower in apparent mol wt than intact FSH, were separated by PAGE, but not by gel filtration chromatography (Sephadex G-25). PAGE purification of radioligand resulted in significantly greater (compared to gel filtration) RRA sensitivity and specificity. Maximum binding of PAGE-purified [125I]hFSH to excess calf tests membrane receptors was 45%, with a specific activity of approximately 26 microCi/micrograms, as determined by the method of self-displacement. Maximum binding to excess hFSH antisera (NIH anti-hFSH 4) was 80-85%. This allowed a useful final dilution of 1:120,000, thereby facilitating development of a sensitive and specific RIA with this antiserum. These data indicate that PAGE separation of intact [125I]hFSH from other iodinated proteins results in improved radioligand binding, assay sensitivity, and assay specificity. In addition, PAGE-purified lactoperoxidase-iodinated hFSH is suitable for use in both RIA and RRA.     

Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.

We have integrated preparative two-dimensional polyacrylamide gel electrophoresis with high-performance tandem mass spectrometry and Edman degradation. By using this approach, we have isolated and identified, by partial sequencing, a human melanoma protein (34 kDa, pI 6.4) as lipocortin I. To our knowledge, this protein was not previously known to be associated with melanoma cells. The identity of the protein was confirmed by two-dimensional immunoblot analysis. High-energy collision-induced dissociation analysis revealed the sequence and acetylation of the N-terminal tryptic peptide and an acrylamide-modified cysteine in another tryptic peptide. Thus, knowledge concerning both the primary structure and covalent modifications of proteins isolated from two-dimensional gels can be obtained directly by this approach, which is applicable to a broad range of biological problems.     

Analysis of inborn errors of metabolism and other genetic defects in human fibroblasts using two-dimensional polypeptide mapping

A standardized technique for the two-dimensional polypeptide mapping of cultured human fibroblasts has been used for the study of cellular protein variations in healthy controls, patients with inborn errors of metabolism and some other genetic defects. The analysis of about 50 gels has established that this method is very reproducible and enables the examination of about 600 polypeptides in a single gel. The inter-individual variation has been rather low (about 3%). In the gels from patients with genetic defects only very minor qualitative variations have been observed.     

Analysis of inborn errors of metabolism and other genetic defects in human fibroblasts using two-dimensional polypeptide mapping

A standardized technique for the two-dimensional polypeptide mapping of cultured human fibroblasts has been used for the study of cellular protein variations in healthy controls, patients with inborn errors of metabolism and some other genetic defects. The analysis of about 50 gels has established that this method is very reproducible and enables the examination of about 600 polypeptides in a single gel. The inter-individual variation has been rather low (about 3%). In the gels from patients with genetic defects only very minor qualitative variations have been observed.     

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.