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Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix.
Gessel M, Spraggins JM, Voziyan P, Hudson BG, Caprioli RM
(2015) J Mass Spectrom 50: 1288-93
MeSH Terms: Amino Acid Sequence, Animals, Collagen, Extracellular Matrix, Extracellular Matrix Proteins, Freezing, Humans, Hydrolysis, Image Processing, Computer-Assisted, Mice, Molecular Sequence Data, Peptide Fragments, Rats, Sodium Dodecyl Sulfate, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry
Show Abstract · Added November 3, 2015
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful molecular mapping technology that offers unbiased visualization of the spatial arrangement of biomolecules in tissue. Although there has been a significant increase in the number of applications employing this technology, the extracellular matrix (ECM) has received little attention, likely because ECM proteins are mostly large, insoluble and heavily cross-linked. We have developed a new sample preparation approach to enable MALDI IMS analysis of ECM proteins in tissue. Prior to freezing and sectioning, intact tissues are decellularized by incubation in sodium dodecyl sulfate. Decellularization removes the highly abundant, soluble species that dominate a MALDI IMS spectrum while preserving the structural integrity of the ECM. In situ tryptic hydrolysis and imaging of tryptic peptides are then carried out to accommodate the large sizes of ECM proteins. This new approach allows the use of MALDI IMS for identification of spatially specific changes in ECM protein expression and modification in tissue.
Copyright © 2015 John Wiley & Sons, Ltd.
1 Communities
4 Members
1 Resources
16 MeSH Terms
The activity of prolactin releasing peptide correlates with its helicity.
Deluca SH, Rathmann D, Beck-Sickinger AG, Meiler J
(2013) Biopolymers 99: 314-25
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, COS Cells, Cell Line, Tumor, Cercopithecus aethiops, Circular Dichroism, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Mutation, Prolactin-Releasing Hormone, Protein Binding, Protein Conformation, Protein Stability, Protein Structure, Secondary, Receptors, Neuropeptide, Signal Transduction, Sodium Dodecyl Sulfate, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Temperature, Trifluoroacetic Acid
Show Abstract · Added January 24, 2015
The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C-terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8-20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5-10-fold loss of activity was found for PrRP8-20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTANMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full-length peptide, which exists in an equilibrium between α- and 3(10)-helix. We demonstrate that PrRP8-20's reduced propensity to form an α-helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α-helical C-terminal region of PrRP is critical for receptor activation.
Copyright © 2012 Wiley Periodicals, Inc.
1 Communities
1 Members
0 Resources
24 MeSH Terms
Letter to the editor. Chemical-genetic strategy for inhibiting proteasome function in Saccharomyces cerevisiae.
Howard GC, Collins GA, Tansey WP
(2012) Yeast 29: 93-4
MeSH Terms: ATP-Binding Cassette Transporters, Cysteine Endopeptidases, DNA, Mitochondrial, Gene Deletion, Genome, Fungal, Leupeptins, Proline, Proteasome Endopeptidase Complex, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sodium Dodecyl Sulfate, Species Specificity
Added March 10, 2014
0 Communities
1 Members
0 Resources
12 MeSH Terms
Coupling of conformational and proteolytic activation in the kinetic mechanism of plasminogen activation by streptokinase.
Boxrud PD, Bock PE
(2004) J Biol Chem 279: 36642-9
MeSH Terms: Catalysis, Dose-Response Relationship, Drug, Fibrinolysin, Hydrolysis, Kinetics, Models, Chemical, Models, Molecular, Plasminogen, Plasminogen Activators, Protein Binding, Protein Conformation, Sodium Dodecyl Sulfate, Streptokinase, Time Factors
Show Abstract · Added January 20, 2015
Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v(1)) representing conformational activation and subsequent rates of activity increase (v(2)) corresponding to the rate of Pm generation determined by a specific discontinuous assay. The v(2) dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of approximately 2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an approximately 12-fold lower affinity for SK and an approximately 20-fold lower k(cat)/K(m). Blocking lysine-binding sites on Pg inhibited SK.Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu]Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK.Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK.Pg* and subsequent cleavage of Pg by SK.Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK.Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK.Pm, which then converts the remaining free Pg to Pm.
0 Communities
1 Members
0 Resources
14 MeSH Terms
NMR studies of the anti-apoptotic protein Bcl-xL in micelles.
Losonczi JA, Olejniczak ET, Betz SF, Harlan JE, Mack J, Fesik SW
(2000) Biochemistry 39: 11024-33
MeSH Terms: Amino Acid Sequence, Apoptosis, Binding Sites, Circular Dichroism, Detergents, Endopeptidases, Humans, Hydrolysis, Micelles, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Phospholipid Ethers, Phosphorylcholine, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Proto-Oncogene Proteins c-bcl-2, Sodium Dodecyl Sulfate, Solutions, Structure-Activity Relationship, Ultracentrifugation, Water, bcl-X Protein
Show Abstract · Added March 5, 2014
The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.
0 Communities
1 Members
0 Resources
23 MeSH Terms
Reductive cleavage of the disulfide bonds of the collagen IV noncollagenous domain in aqueous sodium dodecyl sulfate: absence of intermolecular nondisulfide cross-links.
Reddy GK, Hudson BG, Bailey AJ, Noelken ME
(1993) Biochem Biophys Res Commun 190: 277-82
MeSH Terms: Alkylation, Animals, Aorta, Basement Membrane, Cattle, Chromatography, High Pressure Liquid, Collagen, Cross-Linking Reagents, Disulfides, Dithiothreitol, Electrophoresis, Polyacrylamide Gel, Female, Humans, Hydrolysis, Kidney Glomerulus, Lysine, Macromolecular Substances, Mercaptoethanol, Muscle, Smooth, Vascular, Oxidation-Reduction, Placenta, Pregnancy, Sodium Dodecyl Sulfate
Show Abstract · Added December 10, 2013
The subunits of the collagen IV hexameric, noncollagenous NC1 domain obtained from bovine aorta, glomerular basement membrane, alveolar basement membrane and placental basement membrane are predominantly dimers. A large fraction of the dimers had been thought to be linked by nondisulfide bonds because they were resistant to cleavage by 5% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, at 100 degrees C. However, if an unusually high concentration of 2-mercaptoethanol, e.g., 40% (v/v), is used, complete conversion of dimers into monomers is achieved, indicating the lack of intersubunit nondisulfide cross-links. Electrophoresis patterns indicate that some of the intermolecular disulfide bonds of the dimers are more resistant to reduction in aqueous SDS than are some of the intramolecular disulfide bonds.
1 Communities
1 Members
0 Resources
23 MeSH Terms
Estimation of the size of collagenous polypeptides by sodium dodecyl sulfate--polyacrylamide gel electrophoresis.
Noelken ME, Wisdom BJ, Hudson BG
(1981) Anal Biochem 110: 131-6
MeSH Terms: Animals, Cattle, Collagen, Cyanogen Bromide, Electrophoresis, Polyacrylamide Gel, Molecular Weight, Peptides, Skin, Sodium Dodecyl Sulfate
Added December 10, 2013
1 Communities
1 Members
0 Resources
9 MeSH Terms
Fractionation of glycoprotein components of the reduced alkylated renal glomerular basement membrane.
Hudson BG, Spiro RG
(1972) J Biol Chem 247: 4239-47
MeSH Terms: Alkylation, Amino Acids, Animals, Basement Membrane, Carbohydrates, Cattle, Chemical Phenomena, Chemistry, Chromatography, DEAE-Cellulose, Chromatography, Gel, Disaccharides, Disulfides, Electrophoresis, Glycoproteins, Hexosamines, Hexoses, Hydroxylysine, Kidney Glomerulus, Molecular Weight, Neuraminic Acids, Oxidation-Reduction, Peptides, Polysaccharides, Sodium Dodecyl Sulfate, Urea
Added December 10, 2013
1 Communities
1 Members
0 Resources
25 MeSH Terms
Studies on the native and reduced alkylated renal glomerular basement membrane. Solubility, subunit size, and reaction with cyanogen bromide.
Hudson BG, Spiro RG
(1972) J Biol Chem 247: 4229-38
MeSH Terms: Alkylation, Amino Acids, Animals, Basement Membrane, Carbohydrates, Cattle, Chromatography, Gel, Cyanides, Cyanogen Bromide, Electrophoresis, Hexosamines, Hexoses, Kidney Glomerulus, Mercaptoethanol, Methionine, Molecular Weight, Neuraminic Acids, Oxidation-Reduction, Sodium Dodecyl Sulfate, Solubility, Sulfoxides, Surface-Active Agents, Urea
Added December 10, 2013
1 Communities
1 Members
0 Resources
23 MeSH Terms
Eukaryotic topoisomerase II. Characterization of enzyme turnover.
Osheroff N
(1986) J Biol Chem 261: 9944-50
MeSH Terms: Adenosine Diphosphate, Adenylyl Imidodiphosphate, Animals, Cytidine Triphosphate, DNA Topoisomerases, Type II, DNA, Circular, DNA, Superhelical, Drosophila melanogaster, Electrophoresis, Agar Gel, Kinetics, Magnesium, Magnesium Chloride, Sodium Dodecyl Sulfate
Show Abstract · Added March 5, 2014
While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.
0 Communities
1 Members
0 Resources
13 MeSH Terms