Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.     

Characteristics of thyroid protein kinase C. Different Ca2 requirement for the phosphorylation of endogenous proteins and of H1 histone

Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP-dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities. Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (approximately 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I. The one-dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22-25 kDa and 32-36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins. A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc-specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one-dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins. Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two-dimensional PAGE into several distinct spots with molecular masses of 32-35 kDa and pI range of 5-7.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein Profile of <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> Strains Exhibiting Different Levels of Tolerance to Metal Sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe²⁺ oxidation in the presence of 250 mM of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30–70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and I35) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

Isolation and characterization of a 25-kilodalton protein from mouse testis: sequence homology with a phospholipid-binding protein.

A 25-kDa epididymal secretory protein (MEP 9), isolated from mouse epididymal fluid, has recently been characterized in our laboratory [Rankin et al., Biol Reprod 1992; 46:747-766]. The polyclonal antibody raised against this protein was found to recognize a 25-kDa component in epididymal fluid and testicular extract. The 25-kDa testicular antigen (MTP) was purified by means of ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography; MTP was found to be similar to MEP 9 in several properties including molecular mass (25 kDa), isoelectric point (pI 6.0), and immunoreactivity when the proteins were resolved in the presence of SDS (one-dimensional and two-dimensional PAGE). However, when the proteins were resolved under non-denaturing conditions, MTP showed strong immunoreactivity while MEP 9 did not. This observation suggests that although the 25-kDa antigens from the epididymal fluid and testicular extract are quite similar, they may have different immunological conformations. When analyzed for amino acid composition and partial amino acid sequence, the testicular antigen showed substantial homology (> 80%) with a phosphatidylethanolamine-binding protein characterized from bovine brain. MTP also showed phosphatidylethanolamine-binding activity (Kd = 1.95 x 10(-5) M, Bmax = 1.86 nmol/micrograms MTP), suggesting that the mouse 25-kDa protein is a member of the phospholipid-binding protein family and may have a role in lipid metabolism during sperm maturation.     

Production of novel proteins by Bacteroides ureolyticus grown under conditions of reduced iron availability

Protein profiles of whole cells of Bacteriodes ureolyticus grown in the presence or absence of the iron chelator desferrioxamine mesylate (Desferal) were compared. Each of four strains produced novel proteins of molecular weights 19, 25 and 41 kilodaltons (kDa) under conditions of reduced iron availability. Novel proteins of molecular weights 32, 52 and 58 kDa were also detected although there was interstrain variation in their expression. Outer membranes from three of the strains grown on iron-depleted medium also contained novel proteins with molecular weights of approximately 25, 41 and 52 kDa. When organisms were grown on medium containing Desferal saturated with excess iron, the novel proteins were not detected indicating that their expression was regulated by the level of available iron in the medium.     

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.     

The alteration of mRNA expression of SOD and GPX genes,and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles

Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L⁻¹) and ZnO-NPs (0, 15 and 30 mg L⁻¹). Treatments with NaCl at both 3 and 6 g L⁻¹ suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS–PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.     

Studies on middle silkgland proteins of cocoon colour sex-limited silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) using two-dimensional polyacrylamide gel electrophoresis

Qualitative and quantitative differences in proteins expressed in the middle silkglands of male and female silkworm larvae that differ in silk colour were investigated by high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer assisted image analysis. About 1000 protein spots were resolved in both the sexes and most proteins were shown to be distributed in the area from 15 kDa to 70 kDa and pH 4–8. It was found that some proteins displayed higher expression in yellow cocoon, while two proteins were only expressed in female silkworm silkgland tissue through the comparison and analysis by two-D software. These proteins especially existed in female silkworm middle silkgland tissue of yellow cocoon. Furthermore, these proteins might be involved in the expression of cocoon colour phenotype     

The effectiveness of grafting to improve salt tolerance in tomato when an ‘excluder’ genotype is used as scion

If the main effect of long-term exposure of tomato plants to salinity is the accumulation of toxic concentrations of Na⁺ and Cl⁻ in the leaves, then the selection of ‘excluder’ rootstocks should increase tolerance to salinity in grafted tomato plants, independently of the genotype used as the scion. The question addressed in this study is whether shoot genotypes with an ‘excluder’ character are able to increase their salt tolerance when grafted onto rootstocks of the same characteristics. Moneymaker (with excluder character) was grafted onto two root genotypes, Radja and Pera, selected for their very different ability to regulate the transport of saline ions to the shoot over time. Grafting onto either Pera or Radja improved fruit yield compared to the self-grafted plants of Moneymaker (M/M) when the plants were grown at 50 mM NaCl, whereas there was no effect of either rootstock or of grafting per se (M/M) on fruit yield in the absence of or at 25 mM NaCl. The relationship between the salt responses to mid- and long-term depended on the stress level; after 27 d of 150 mM NaCl treatment, both graft combinations enhanced similarly their salt tolerances as did in the long-term experiment. Moreover, the tolerance induced by rootstock was related to the low rates of saline ion accumulation in their leaves. However, the positive effect of rootstock was only observed with rootstock Pera when the grafted plants were grown at 50 mM NaCl (the same salt level used in the long-term experiment) for 35 d. According to the physiological changes induced by rootstock in the leaves, the different salt responses seem to be due to the fact that the osmotic effect predominated on the toxic effect under these last conditions. Consequently, in order to select rootstocks care must be taken in the timing of any selection process: the stress level and length of exposure to salinity must be sufficient for the true differences in salt tolerance for toxicity to be shown. Taken together, these results show the effectiveness of grafting to enhance fruit yield in tomato and provide evidence that the positive effect induced by rootstock is related to the re-establishment of ionic homeostasis.     

Mammalian FKBP-25 and its associated proteins

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.     

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS–PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS–PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.     

Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.     

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

多种小麦种子储藏蛋白的电泳分析

采用不同的提取液,对１０个小麦品种的非酶功能性种子储藏蛋白进行提取,分别进行梯度凝胶电泳分析。电泳依据提取液的不同,分别采用酸性或碱性系统。对酸性凝胶催化系统,采用Ａｐ－Ｖｃ－ＦｅＳＯ４系统代替Ｈ２Ｏ２－Ｖｃ－ＦｅＳＯ４系统,克服了酸性凝胶的不足,提高了凝胶的性质性能并使之容易操作。应用新的催化系统配制的酸性梯度胶,提高了分辨率。并初步尝试以酸性系统分析种子谷蛋白,获得了成功,经过对不同提取液蛋白     

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.