Urea Gel Electrophoresis Research Articles

Protamines and the sperm nuclear basic proteins Pandora's Box of insects.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

Characterization of a 36 kDa antigenic protein of fish-specific monoclonal-antibody 8F5

The 36 kDa antigenic protein of a fish-specific monoclonal antibody (mAb), 8F5, previously developed to detect fish in foodstuffs to protect fish allergic individuals, was characterized to establish its identity and to identify the fish-specific epitope. We hypothesized that this antigenic protein is tropomyosin based on its thermal stability and molecular weight. Western blot showed that both the 36 kDa protein and fish tropomyosin were recognized by mAb 8F5, and their molecular weight migration in urea gel electrophoresis was identical. In addition to matching the amino acid composition profile, this 36 kDa protein’s sequences precisely correspond to those in fish tropomyosin fragments. Further analysis revealed the sequence of the fish-specific epitope bound by mAb 8F5 to be EDDLVALQKK. These results confirm that the 36 kDa protein is indeed tropomyosin and will be a suitable biomarker for the immunodetection of fish in cooked food.

Urea Gel Electrophoresis in Studies of Conformational Changes of Transferrin on Binding and Transport of Non-Ferric Metal Ions.

Transferrin (Tf) is a crucial transporter protein for Fe(III), but its biological role in binding other metal ions and their delivery into cells remain highly controversial. The first systematic exploration of the effect of non-Fe(III) metal ion binding on Tf conformation has been performed by urea-polyacrylamide gel electrophoresis (urea-PAGE), which is commonly used for nucleic acids but rarely for proteins. Closed Tf conformation, similar to that caused by Fe(III)-Tf binding, was formed for In(III), V(III) or Cr(III) binding to Tf. In all these cases, metal distribution between Tf lobes and/or the rate of metal release under acidic conditions differed from that of Fe(III)-Tf. By contrast, Ga(III) and V(IV) did not form closed Tf conformation under urea-PAGE conditions. Apart from Fe(III), only In(III) was able to increase the proportion of closed Tf conformation in whole serum. These results suggest that Tf is unlikely to act as a natural carrier of any metal ion, except Fe(III), into cells but can reduce toxicity of exogenous metal ions by binding them in serum and preventing their entry into cells.

A COMPETITIVE EX-VIVO SCREENING ASSAY TO DETERMINE ABETA ANTIBODY MEDIATED PHAGOCYTOSIS AND PLAQUE SPECIFICITY

Extracellular amyloid plaque is one of the pathological hallmarks of Alzheimer's disease (AD). Up to 75% of the insoluble Aβ in AD brain is N-terminally truncated or modified, including Aβp3–42, which has only been detected in plaque. We reported previously the development of a monoclonal antibody that specifically targets Aβp3–42. The antibody showed robust clearance of pre-existing plaque without causing microhemorrhage (DeMattos et al, Neuron 2012). The Aβp3–42 antibodies with either minimal (IgG1 isotype) or maximal (IgG2a) effector function strongly labeled deposited plaque in both AD and PDAPP brain sections and led to significant reduction of Aβ1–42 in ex vivo phagocytosis assay. For screening and identification of future plaque-specific antibodies, we developed a functional antibody mediated phagocytosis assay to identify antibodies that would not cross react with full length Aβ and potentially be saturated by the soluble pool. We examined plaque specificity of Aβ antibodies by pre-incubating soluble Aβ1–40 with the antibodies prior to ex vivo phagocytosis assay with AD brain sections and murine microglia. Amyloid plaques were then fully solubilized and reduced to monomers by formic acid and Aβ peptides were resolved by acid urea gel electrophoresis coupled with western blotting analysis. Changes in distinctive Aβ species were then quantified. Using this method, we examined the plaque specific humanized Aβp3–42 antibody as well as the comparator 3D6 antibody which recognizes plaque as well as soluble Aβ. Consistent with our previous finding with murine Aβp3–42 antibody, humanized Aβp3–42 antibody significantly enhanced Aβ clearance via microglia-mediated phagocytosis. This effect was not influenced by pre-incubation of the antibody with soluble Aβ1–40 peptide, as revealed by acid urea gel electrophoresis, reflecting the plaque-specific characteristics of Aβp3–42 antibody. Humanized 3D6 antibody also invoked significant Aβ clearance. However, this effect was largely blocked by soluble Aβ1–40, reflecting the non-plaque specific nature of 3D6. We have developed an ex vivo functional assay for evaluating plaque specificity of Aβ antibodies. The assay will facilitate screening of future generation antibodies that target amyloid plaque.

A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2.

The exchange of histones for transition proteins (TNPs) and finally protamines is an essential process during spermatogenesis that enables the strong condensation of chromatin during sperm formation. Research on this process obviously depends on the availability of specific antibodies recognizing these nuclear proteins. A commercial antibody generated against human protamine-2 (PRM2) has been described to cross-react with mouse PRM2 and in fact has been used in several studies to detect mouse PRM2. Some inconsistent results obtained with this goat-derived antibody prompted us to re-examine its specificity. In immunofluorescence experiments with epididymal sperm, only a low percentage of sperm nuclei were stained by this antibody, whereas a mouse monoclonal anti- PRM2 antibody stained most sperm, as expected. Western blot analysis of basic nuclear proteins from spermatids and sperm separated by acid urea (AU) gel electrophoresis revealed that the goat anti- PRM2 antiserum binds to mouse TNP2 but not mouse PRM2. Epitope mapping using glutathione-S-transferase-fusion proteins with peptide sequences conserved in human PRM2 and mouse TNP2 identified the tetrapeptide arginyl-lysyl-arginyl-threonine as an epitope of the goat anti- PRM2 antiserum. Our findings underline the importance of using AU gel electrophoresis to confirm specificities of antibodies directed against basic nuclear proteins, which are not well separated, and may show abnormal migration behaviour, in SDS-polyacrylamide gel electrophoresis.

Cu2+-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants

Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu2+ with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton–acid–urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu2+ chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu2+ ions.

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05). A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage. Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE. No personal or direct financial support has been received for any of this work. The authors declare no competing interests. N/A.

Interaction of vanadium(IV) with human serum apo-transferrin

The interaction of VIVO-salts as well as of a few VIVO(carrier)n complexes with human serum transferrin (hTF) is studied focusing on the determination of the nature and stoichiometry of the binding of VIVO2+ to hTF, as well as whether the conformation of hTF upon binding to VIVO2+ or to its complexes is changed. Circular dichroism (CD) spectra measured for solutions containing VIVO2+ and apo-hTF, and VIVO–maltol and apo-hTF, clearly indicate that hTF–VIVO–maltol ternary species form with a VIVO:maltol stoichiometry of 1:1. For VIVO salts and several VIVO(carrier)n complexes (carrier ligand=maltolato, dhp, picolinato and dipicolinato) (Hdhp=1,2-dimethyl-3-hydroxy-4-pyridinone) the maximum number of VIVO2+ bound per mole of hTF is determined to be ~2 or lower in all cases. The binding of VIVO to apo-hTF most certainly involves several amino acid residues of the Fe-binding site, and as concluded by urea gel electrophoresis experiments, the formation of (VIVO)2hTF species may occur with the closing of the hTF conformation as is the case in (FeIII)2hTF, which is an essential feature for the transferrin receptor recognition.

Independent Generation of Aβ42 and Aβ38 Peptide Species by γ-Secretase

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.

Amino acid sequence and phosphorylation status of myosin light chain MLC20 during vascular smooth muscle cell maturation

Myosin light chain kinase (MLCK) is an important enzymatic regulator of vascular contractility that phosphorylates the regulatory protein, myosin light chain 20 (MLC20). We have recently demonstrated that the specific activity of MLCK is strikingly greater in fetal vs. adult sheep carotid arteries. Because cell‐specific splicing isoforms of myosin‐related genes have been identified in vascular smooth muscle, defining a late stage in the differentiation pathway from the synthetic to contractile phenotype, we tested the hypothesis that developmental alteration in MLCK activity may be at least partially dependent on developmental regulation of MLC20 isoforms. We also tested the hypothesis that developmental differences in baseline phosphorylation of MLC20 may influence MLCK activity. MLC20 was sequenced using a combination of biochemical purification and electron‐spray ionization‐mass spectrometry (ESI‐MS). Phosphorylation of MLC20 was quantified by urea gel electrophoresis and immunoblotting with antibodies against both total MLC and SER19‐phospho‐MLC. Our results demonstrate a high level of amino acid sequence conservation in both N‐terminal and C‐terminal regions of sheep MLC20 in comparison to other mammalian species and are consistent with the hypothesis that developmental shifts in baseline phosphorylation influence MLCK activity. Supported by NIH grants HD31226, HL54120, and HL64867

Biochemical and spectroscopic studies of human melanotransferrin (MTf): Electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6 M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

Probing the stability of native and activated forms of α 2-macroglobulin

α 2-Macroglobulin (α 2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ∼40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α 2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α 2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α 2M complexes.

Extraction, purification and analysis of histones

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.

Biochemical Characterization of VlmL, a Seryl-tRNA Synthetase Encoded by the Valanimycin Biosynthetic Gene Cluster

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Baseline Nucleophosmin Status in Mutated(M) Versus Unmutated (U) Immunoglobulin B-CLL Is a Nuclear Reflection of Different Signal Transduction Physiology.

Aim: The difference in prognosis between B-CLL patients with (M) and wihout (U) somatic hypermutations of the immunoglobulin genes has led to a search for surrogate markers in routine practice. cDNA-Microarray differentiation between M and U led to further analysis of Zap-70, fibromodulin and LPLase, all of which succesfully discriminate the majority of M vs U.. In 1 of 2 proteomic studies searching the difference between M and U B-CLL, nucleophosmin came out as uniformly absent in 6 U patients and present in all 6 with M B-CLL. Nucleophosmin is a nucleolar non-ribosomal protein, associated with cellular proliferation. It is implicated in carcinogenesis as a differentiation blocker in K562 myeloid leukemias where its degradation from a 35 to a 21 kD form accompanies phorbol ester induced differentiation. In K562 this degradation is dependent on P-Erk-translocation to the nucleus that by itself depends on prolonged Erk-activation. We have recently demonstrated that SDF-1, an important microenvironment factor in B-CLL, is capable of inducing prolonged Erk-activation in Zap-70+ but not in Zap-70- B-CLL cells in vitro. This prompted us to study the unstimulated expression of nucleophosmin and other nuclear proteins by Westen blot in 16 patients with B-CLL.Methods: Histones and other alkalic proteins were extracted from the nuclei (pellet fraction) upon treatment with 0.4 M HCl. Following precipitation in acetone, histones and proteins were resuspended in sample buffer and separated by SDS PAGE or acid urea gel electrophoresis. Subsequently, the proteins were electrotransferred to a nitrocellulose membrane. Western blot analysis was performed against nucleophosmin, histone H3 or H4, and final signal detection was revealed by enhanced chemiluminescence reaction.Results: The 35 kD nucleophosmin signal was uniformly expressed in all samples and related to total histone content, irrespective of mutation status, cytogenetics and stage of disease. However, a minor band at 21 kD reflecting degradation was present in 6/6 patients with U B-CLL and absent in 8/10 with M B-CLL. Pan-histone H3 and H4 antibodies revealed similar banding patterns for histone H3 in all patients, but the presence of a heavier (probably ubiquinated) double band for histone H4 in some. This ubiquinated fraction was important in 7/16, faint in 5/16 and absent in 4/16. Morphology was atypical in 4/7 with an important fraction, and typical in all others. 5/7 with important bands and 4/5 with faint bands were M B-CLL against only 1 out of 4 in the group with absence of ubiquitination bands.Discussion: In contrast to previous proteomic studies, we found the 35 kD form of nucleophosmin present in both M and U B-CLL. However, nucleophosmin metabolisation to the 21 kD form is observed preferentially in the U B-CLL. The increased baseline degradation of nucleophosmin in 6/6 U B-CLL all of whom are Zap-70+ can be explained as a downstream nuclear signal from constitutive increased Erk-phosphorylation upon environmental signals in Zap-70+ B-CLL.

Non‐transferrin‐bound iron in platelet concentrates promotes the growth of Staphylococcus epidermidis

Staphylococcus epidermidis, the most common organism implicated in bacterial contamination of platelet (PLT) concentrates (PCs), does not grow in serum unless transferrin is fully saturated and there is non-transferrin-bound iron (NTBI) available. Here, the occurrence and origin of NTBI in PCs has been studied. NTBI in PC supernatants was determined by a chelation method and by the bleomycin-detectable iron assay. Iron binding by transferrin was determined by spectrophotometry, and transferrin iron forms, by urea gel electrophoresis. The growth of inoculated S. epidermidis in PC supernatants was monitored by optical density and determination of viable counts. PCs contained approximately 0.14 micromol per L redox-active iron measured by the bleomycin assay and approximately 0.7 micromol per L NTBI by the chelation method. As a further indication of the presence of NTBI, the growth of S. epidermidis in the PC supernatants was inhibited by iron chelation with deferoxamine. Transferrin in the PC medium was only partially saturated with iron, and the reason for the presence of NTBI was found to be impaired iron binding by transferrin. Iron was displaced from transferrin by citrate at molar ratios to transferrin that occur in citrated plasma and in PLT additive solution (AS). Citrated plasma supported the growth of S. epidermidis whereas serum did not. PCs stored in plasma or AS contain a low level of NTBI because of the displacement of iron from plasma-derived transferrin by citrate. NTBI in the PC medium supports the growth of S. epidermidis.

RNA Structural Rearrangement via Unwinding and Annealing by the Cyanobacterial RNA Helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.

Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants.

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).

Purification of recombinant plasminogen activator inhibitor-1 in the active conformation by refolding from inclusion bodies

Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators. Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions. Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity. In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells. The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction. Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1. The purified protein yielded a single, 43 kDa protein band upon SDS–polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources. Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions. Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation.