Two-dimensional SDS-polyacrylamide Gel Research Articles

Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

Potential mechanism of apoptosis induced by ultrasound in human hepatocarcinoma cells via comparative proteomic analysis.

To analyze the potential molecular mechanism of ultrasound induced apoptosis in cancer cells, comparative proteomic methods were introduced in the study. After ultrasound exposure at the intensity of 1.2 W/cm2, the human SMMC-7721 hepatocarcinoma cells were stained by trypan blue to detect the morphologic changes, and then the flow cytometry was used to examine the percentage of early apoptosis via double staining of FITC-labelled Annexin V and Propidium iodide. The proteins were separated by two-dimensional (2D) SDS polyacrylamide gel electrophoresis (PAGE). Among them, the differently expressed proteins were identified by MALDI-TOF mass spectrometry to reveal the key proteins response to ultrasound exposure. It's proved early apoptosis of cells were induced by focused ultrasound. After ultrasound exposure, the expressing characteristics of several proteins changed, in which some proteins in HSP family are associated with apoptosis initiation. It is suggested that the focused ultrasound could be applied in the assistant cancer therapy. Moreover, it is proved the comparative proteomic methods could supply information about the protein expression to analyze the metabolic processes related to bio-effects of biomedical ultrasound.

A shift in erythrocyte histone H1 complement following selection in quail (Coturnix japonica)

This work was aimed at comparing distribution of isoforms for polymorphic histone H1 variants H1.b and H1.z and variably abundant histone H1.d subtype between quail ( Coturnix japonica ) population selected for a high egg yolk cholesterol content and the control birds. The isoforms of histone H1.b (H1.b1, H1.b2) and histone H1.z (H1.z1, H1.z2) differed in their apparent molecular weights judging from their differential migration rates in one- and two-dimensional SDS-polyacrylamide gels. Stained histone H1.d bands and spots in one-dimensional acetic acid-urea and two-dimensional SDS-polyacrylamide gel patterns, respectively, exhibited differential intensities among quail individuals. Histone H1.d phenotypes with high (d h ), intermediate (d h d l ), and low (d l ) amount of protein, displaying a small within-phenotype variability of the protein band intensities (coefficients of band variation below a threshold value of 0.25) were shown to be inherited in a Mendelian fashion with two alleles at a locus contributing to the band intensity. The frequencies of histone H1 alleles at loci H1.b (χ 2 = 13.32, d.f. = 1, P < 0.001), H1.z (χ 2 = 21.84, d.f. = 1, P < 0.001), and H1.d (χ 2 = 8.98, d.f. = 1, P < 0.01) were found to be statistically significant among the control and selected population. In general, a moderate degree of genetic divergence (F ST equal to 0.07 and 0.1 at loci H1.b and H1.z, respectively) was observed among the control and selected quail populations. Selection may directly or indirectly affect the complement of H1 histones because of their presumably differential interactions with DNA and/or DNA-associated proteins resulting in alterations in the chromatin function.

Proteome Analysis of Phenol-Degrading Achromobacter sp. Strain C-1, Isolated from an Industrial Area

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, Achromobacter sp. strain C-1, from an industrial area (Cubatão, Brazil). Achromobacter sp. is a non-motile, strictly aerobic, gram-negative, short-rod or coccobacillary bacterium, which can occur singly, in pairs or in clusters. 16S rRNA gene sequence analysis revealed that Achromobacter sp. strain C-1 belongs to the gamma group of Proteobacteria, with 99% similarity to 16S rRNA gene sequences of Achromobacter xylosoxidans. Strain C-1 can grow aerobically on a number of aromatic compounds, such as phenol, catechol, m-cresol and o-cresol. In particular, it can degrade up to 600 mg/L of phenol at 37°C. In order to understand the phenol degradation pathway used by strain C-1, phenol or glucose cultured C-1 proteomes were comparatively analyzed with two-dimensional SDS-polyacrylamide gel (2D SDS PAGE). Nine protein spots were exclusively induced from phenol-cultures of strain C-1. Of these 9 spots, three phenol-degrading enzymes (phenol degradation meta-pathway protein, hydroxymuconic semialdehyde dehydrogenase and 4-hydroxy-2-oxovalerate aldolase) were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides, which suggests that strain C-1 degrades phenol via the β-ketoadipate pathway. Analysis of the metabolite produced from phenol proved that this enzyme is a catechol 2,3 dioxygenase, which is able to utilize phenol as a substrate. These results suggest that comparative proteomic analysis of biodegrading bacteria cultures under different conditions may be a useful initial step toward the elucidation of an aromatic compound degradation pathway and the strain C-1 could be an excellent candidate for the biotreatment of phenol-containing industrial wastewaters.

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site

We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

A methodical approach for improving the reliability of quantifiable two-dimensional Western blots

Western transfer after the electrophoretic separation of proteins onto an adsorbent membrane, with subsequent immunodetection, is a powerful tool for detecting and characterizing a multitude of proteins. An important aspect of the study of proteins is that they often exist as isoforms with structural microheterogeneity giving rise to differences in biological activity. Western blotting (WB) in combination with two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) allows the specific quantification of single isoforms of a protein. We have investigated whether a methodical modification of 2D-SDS-PAGE improves the quality of quantifiable 2D-WB data. The effect of a combined separation of three previously electrofocused protein extracts lying side by side on a single SDS-gel in parallel has been tested against the traditional procedure, viz., the separation of one protein extract per SDS-gel. The modified procedure results in a more reliable and better quality data than the traditional procedure, which seems to be prone to producing systematic or random errors. Our simple practical procedure improves immunoblotting accuracy by excluding numerous sources of errors and saves time, immunoblotting reagents and costly antibodies.

Abstract 3264: Mesothelin expression in ovarian tumors and serum autoantibodies of the laying hen model is similar to human ovarian cancer

Abstract Background: Mesothelin (MSLN) is a 40kDa cell-surface differentiation antigen that is normally expressed at low levels and is restricted to tissues such as the mesothelial cells lining some body cavities and epithelial cells of kidney, tonsil, trachea, and fallopian tubes. However MSLN is highly expressed in ovarian cancer, mesotheliomas, and to a lesser extent in other cancers such as pancreatic, lung, and stomach. Soluble MSLN is shed into the circulation and is a potential serum biomarker for ovarian cancer. In addition MSLN autoantibodies are detected in the sera of patients whose tumors were positive for MSLN. The laying hen spontaneously develops progressive ovarian tumors. Ovarian adenocarcinoma in the hen bears a striking histological resemblance to human epithelial ovarian cancer. Furthermore, multiple proteins are similarly expressed in hen and human ovarian tumors. In this study, we hypothesized that MSLN expression is increased in hen ovarian tumors compared to normal ovaries, and that similar to human ovarian cancer, there are circulating anti-MSLN antibodies in response to tumors. Materials and Methods: 31 White Leghorn laying hens were used for sample collection. MSLN mRNA expression in hen ovarian tumors (n=21) and normal ovaries (n=10) was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). MSLN protein expression was evaluated by immunohistochemistry (IHC) and two-dimensional SDS-Polyacrylamide Gel Electrophoresis (2D-SDS-PAGE) Western blot. Circulating anti-MSLN autoantibodies were assessed by immunoassay of sera from hens with normal ovaries (n=10) and with ovarian tumors (n=10). Results: RT-PCR showed 57% (12/21) of ovarian cancers expressed MSLN. MSLN expression was observed in 3/4 (75%) serous carcinomas, 2/6 (33%) mucinous carcinomas, 1/4 (25%) endometrioid tumors and 6/7 (86%) mixed histology tumors. By tumor stage, 5/8 (62.5%) early stage (I/II) and 7/13 (53.8%) late stage (III/IV) tumors had MSLN expression. None of the normal ovaries had detectable levels of MSLN mRNA. A significant difference of MSLN mRNA expression was seen between tumor and normal (P&amp;lt;0.01), while differences between stage or tumor types were not statistically significant (P&amp;gt;0.05). By IHC and 2D Western blot, immunoreactive MSLN protein was detected in tumors with detectable MSLN mRNA, but not in normal ovaries and tumors without detectable MSLN mRNA. Circulating anti-MSLN antibodies occurred in 60% (6/10) of hens with ovarian tumors. Conclusion: MSLN gene and protein expression is elevated in chicken ovarian tumors and not detectable in normal ovaries. The results support the utility of the hen model for preclinical studies of MSLN as a biomarker and target of therapy. Supported by DOD OC073325 (JL) and NIH R01CA134487 (IH &amp; JL). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3264.

Analysis of Low Molecular Weight Proteome fromH. pyloriCell Extract Using the High Performance Liquid Chromatography

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.

Ligand Binding Rapidly Induces Disulfide-dependent Dimerization of Glycoprotein VI on the Platelet Plasma Membrane

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

Isolation of tissue layers in hermatypic corals by N-acetylcysteine: morphological and proteomic examinations

Corals are diploblastic in body pattern and include two tissue layers, the epidermis and gastrodermis, interconnected by an acellular matrix mesoglea. During development, cells in these tissue layers differentiate morphologically and functionally. In most hermatypic corals, the gastrodermis further develops an ability to associate with microalgae dinoflagellates. This endosymbiosis occurs inside specific host gastrodermal cells, and its mechanism still remains unclear notwithstanding decades of research. The delay in progress is partly due to the difficulty in separating the gastrodermis and its symbionts from the epidermis for detailed cellular and biochemical investigations. The present study reports a simple method to separate these two tissue layers in hermatypic corals using the reducing agent, N-acetylcysteine (NAC). Efficient tissue and proteomic isolations are demonstrated by microscopy and two-dimensional SDS polyacrylamide gel electrophoresis (2D SDS-PAGE). The NAC treatment was able to separate tissue layers without inducing protein degradation. Furthermore, the sensitivity of protein detection greatly increases in the isolated tissue layers. The application of the present technique provides future research on endosymbiosis and coral development with a tool for higher accuracy and sensitivity.

A Glutathione S-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with S-Glutathionylation of Proteins

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

Toward Defining the Human Parotid Gland Salivary Proteome and Peptidome: Identification and Characterization Using 2D SDS−PAGE, Ultrafiltration, HPLC, and Mass Spectrometry

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.

Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying

The aim of this study was to optimize survival of Lactobacillus delbrueckii subsp. bulgaricus during spray-drying and subsequent storage through optimizing the pH of growth conditions. Cell concentrates previously grown without or with pH controlled were spray-dried and stored at 20 degrees C and heat treated at 57 degrees C. Cells grown under noncontrolled pH were more resistant to both drying and heating than cells grown under controlled pH but no significant differences were observed during storage. The intracellular proteins profile of cells grown under both conditions was studied by two-dimensional SDS-polyacrylamide gel electrophoresis. Eight proteins were identified using automated mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data acquisition. Of the identified proteins, only cochaperonin GroES corresponded to a known heat shock protein (HSP). The other proteins identified are proteins involved in glycolysis. For cells grown under noncontrolled pH the expression of the Hsp70, GroES and GroEL, measured by Western blotting, was enhanced. The higher resistance of cells grown under noncontrolled pH correlates with the enhanced production of heat shock proteins. Growth of L. bulgaricus under controlled pH (commonly used by the starter cultures production industry) results in cells more sensitive to stresses frequently encountered by the cells during starter cultures preparation/storage/utilization.

Differential increases in syntheses of newly identified trypsinogen 2 isoforms by dietary protein in rat pancreas.

We have found that dietary protein markedly induced pancreatic serine protease activity via a mechanism independent of luminal trypsin activity in pancreaticobiliary-diverted (PBD) rats. The aim of this study was to examine the effects of dietary protein on the synthesis of trypsinogen isoforms by comparing in vivo incorporation of [35S] L-methionine into isoform proteins in PBD and sham-operated rats. A small duodenal segment including the ampulla of Vater was sectioned and transposed to the upper ileum with end-to-side anastomosis (PBD) or duodenal transection was followed by reanastomosis (sham) in male Sprague-Dawley rats. After recovery, PBD and sham rats were fed a 25% or 60% casein-sucrose-based diet (NC or HC) for 14 days. Rats were then intravenously injected with [35S] L-methionine (15 MBq/kg body weight) 30 mins before being sacrificed for analysis of pancreatic enzymes by two-dimensional SDS-polyacrylamide gel electrophoresis. By using electrophoresis with narrow range of isoelectric focusing (pI 4.5-5.5), five trypsinogen 2 (2-x) isoform spots were identified using both [35S] incorporation and Coomassie brilliant blue (CBB) staining in PBD rats, but not in sham rats. N-terminal sequences of these trypsinogen 2-x spots were identical to known rat trypsinogen 2 with the exception that the third valine was changed to isoleucine in one isoform. In PBD rats, feeding of HC specifically increased the [35S] and CBB intensities of these trypsinogen 2-x isoforms and trypsinogen 3. The degree of induction of the five trypsinogen 2-x molecules by HC varied greatly. Trypsinogen 1 and 4, which are the major trypsinogens in normal rats, showed no changes. We conclude that increases in synthesis of a few newly identified trypsinogen 2-x isoforms mainly contribute to the induction of trypsin activity in the pancreas by HC in PBD rats.

Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary

Post-transcriptional mechanisms play a major role in regulating luteinizing hormone (LH) receptor mRNA expression in the ovary. An ovarian cytosolic protein that we have identified in rats and humans, which binds to a polypyrimidine-rich bipartitate sequence in the coding region of LHR mRNA, acts as a trans-acting factor in this process. In the present study, we isolated and characterized this LH receptor mRNA-binding protein (LRBP) from rat ovary. LRBP was purified to homogeneity by cation exchange chromatography followed by Northwestern analysis and subsequent elution of the single protein band from SDS-polyacrylamide gel. Purified LRBP was subjected to N-terminal microsequencing followed by homology search, which revealed its identity as mevalonate kinase. Purified rat mevalonate kinase antibody recognized the gel-purified LRBP on Western blots performed with one- and two-dimensional SDS-polyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells (293 cells) was tested, it showed all of the characteristics of LRBP with respect to specificity of LHR mRNA binding sequence, as examined by gel mobility shift analysis. Inhibition of LHR mRNA binding activity of mevalonate kinase in the presence of ATP and mevalonate indicates that the RNA recognition site of mevalonate kinase might involve the ATP/mevalonate binding region of the protein. Treatment of 293 cells with mevastatin to deplete cellular mevalonate resulted in an increase in LHR mRNA binding activity of mevalonate kinase. Collectively, the data support the novel function of rat mevalonate kinase as a LHR mRNA-binding protein in the post-transcriptional regulation of LH receptor expression in the ovary.

Proteomic Analysis of Macrophage Differentiation: p46/52Shc TYROSINE PHOSPHORYLATION IS REQUIRED FOR CSF-1-MEDIATED MACROPHAGE DIFFERENTIATION

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.

Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis

Proteins of selected embryonic stages were metabolically labeled with [ 35S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72 kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat ( Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.

Regulation of NADPH-cytochrome P450 reductase expressed during Douglas-fir germination and seedling development.

NADH-cytochrome P450 is a key enzyme that transfers electrons from NADPH to the cytochrome P450 family of enzymes. To begin to determine the regulation of CPR gene expression and enzyme activity in Douglas-fir a full-length cDNA was isolated from a seedling lambda ZAP cDNA library and the ORF was used to develop a synthetic CPR-peptide-based antiserum. Northern blot analysis indicated CPR expression was regulated both developmentally prior to seed maturation and during germination, and differentially in the cotyledons, radicle and megagametophyte of seed and seedling tissues. The CPR-peptide antiserum detected a single CPR in seed and seedling microsomes analyzed by western blot of two-dimensional SDS-polyacrylamide gels. In microsomal extracts from whole seeds and seedlings, the amount of CPR protein remained constant while NADPH:cytochrome c reductase activity increased during stratification, germination and early seedling development. In contrast to cotyledons and megagametophyte, the level of CPR protein detected in radicles was higher than expected when compared to the amount of CPR transcript.