Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.

The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar.

Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level.

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra.

Supercritical fluid extraction of sulphamethazine and its metabolites from meat tissues.

An investigation is reported of factors affecting the supercritical fluid extraction of sulphamethazine and five of its metabolites from spiked meat (swine liver and kidney). The addition of the polar modifier methanol to the carbon dioxide extracting fluid was found to generally enhance recoveries under subcritical and supercritical conditions. Recoveries of the ionic metabolites were increased by up to 72% when employing tetramethylammonium hydroxide for ion pairing in situ with the supercritical fluid extraction.

Analysis of naturally processed peptides eluted from HLA DRB1*0402 and *0404.

Understanding the structural features of naturally processed peptides found within the major histocompatibility complex (MHC) class II peptide binding groove from disease-associated MHC molecules may provide insights into the nature of potential disease-related antigens. Class II MHC/peptide complexes were purified by immunoaffinity from transformed B cell lines homozygous for DRB1*0404 (an allele associated with rheumatoid arthritis) and *0402 (a closely related allele not associated with this disease). Peptides were eluted at acidic pH, fractionated by reversed phase HPLC, and analyzed by capillary electrophoresis.

Future prospects for the analysis of complex biological systems using micro-column liquid chromatography-electrospray tandem mass spectrometry.

An overview is provided of methods for the study of complex biological processes by using micro-column liquid chromatography-electrospray ionization tandem mass spectrometry. Procedures discussed include electrospray ionization, micro-column liquid chromatography, tandem mass spectrometry, tandem mass spectra data interpretation for peptides, and database searching with mass spectral data. Several problems in immunology are discussed to illustrate this approach.

The kinetics of MHC class I and class II expression in rat renal allografts.

We have used in vivo localization of radiolabeled antibodies in a rat renal transplant model to compare the level of induction of major histocompatibility complex (MHC) class I and class II molecules in grafts undergoing rejection with grafts in which rejection was modified by cyclosporine (CsA). MHC class II expression increased in rejecting grafts, peaking on day 4, whereas a later rise in CsA-treated grafts was noted. The use of donor-specific antibodies demonstrated that this was due, in part, to a rise in class II of donor origin.

Binding of sulphamethazine to pig plasma proteins and albumin.

The interaction of sulphamethazine (SMZ) with pig plasma proteins and albumin was studied by ultrafiltration and equilibrium dialysis. Binding to pig plasma proteins was monophasic (affinity approximately 9.0 mol/L x 10(3)) and the main binding protein was albumin.

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus.

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer.

Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes.

The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum.

Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases.

The correlation of uninterpreted tandem mass spectra of modified and unmodified peptides, produced under low-energy (10-50 eV) collision conditions, with nucleotide sequences is demonstrated. In this method nucleotide databases are translated in six reading frames, and the resulting amino acid sequences are searched "on the fly" to identify and fit linear sequences to the fragmentation patterns observed in the tandem mass spectra of peptides. A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted by amino acid sequences translated from the nucleotide database and the fragment ions observed in the tandem mass spectrum.

Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-CgTx GVIA.

We describe a magnetophoretic method for the affinity purification of synaptosomes expressing omega-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of omega-CgTx GVIA which retains its ability to displace [125I] omega-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between omega-CgTx GVIA and the biotin:streptavidin bead complex is sufficiently long to allow flexibility of the toxin to bind to its receptor on synaptosomes.

Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.

A method to correlate uninterpreted tandem mass spectra of modified peptides, produced under low-energy (10-50 eV) collision conditions, with amino acid sequences in a protein database has been developed. The fragmentation patterns observed in the tandem mass spectra of peptides containing covalent modifications is used to directly search and fit linear amino acid sequences in the database. Specific information relevant to sites of modification is not contained in the character-based sequence information of the databases.

Localization of the disulfide bond involved in post-translational processing of glycosylasparaginase and disrupted by a mutation in the Finnish-type aspartylglycosaminuria.

The heavy chain of human glycosylasparaginase (N4-(beta-N-acetylglucosaminyl)-L-asparaginase (EC 3.5.1.

Invariant-cognate peptide exchange restores class II dimer stability in HLA-DM mutants.

Class II presentation mutants have mutations in the HLA-DMA or B genes and are defective in the presentation of whole exogenous Ags restricted by HLA-DR, -DQ, and -DP. The functional defect in Ag presentation is accompanied by an altered conformation of cell surface class II molecules and instability of extracted class II dimers in SDS-PAGE; the latter can be corrected by incubation of mutant cells in an acidic pH in the presence of cognate peptide. Here we investigated the basis for correction of class II dimer instability by acid/cognate peptide treatment and the extent to which this treatment corrects the class II conformational defect in DMB mutants.

An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.

A method to correlate the uninterpreted tandem mass spectra of peptides produced under low energy (10-50 eV) collision conditions with amino acid sequences in the Genpept database has been developed. In this method the protein database is searched to identify linear amino acid sequences within a mass tolerance of ±1 u of the precursor ion molecular weight A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted from amino acid sequences obtained from the database and the fragment ions observed in the tandem mass spectrum. In general, a difference greater than 0.

pH-metric log P. 4. Comparison of partition coefficients determined by HPLC and potentiometric methods to literature values.

The pKa and log P of 20 compounds, including six substituted phenols, two substituted quinolines, N-methylaniline, five barbiturate derivatives, two phenothiazines, and several other molecules of pharmaceutical interest, were determined by the potentiometric technique at 25 degrees C and ionic strength 0.1 M (KNO3). The log P values were determined also by partition HPLC.

Absorption of N4-D-glucopyranosylsulphamethazine by rat everted intestinal sacs.

Absorption of the N4-D-glucose conjugate of sulphamethazine (glucose-SMZ, 0.5 mM) by isolated everted sacs of the rat small intestine was studied at 37 degrees and pH 6.6.

Fragmentation of protonated peptides: surface-induced dissociation in conjunction with a quantum mechanical approach.

This paper describes the results of a systematic investigation designed to assess the utility of surface-induced dissociation in the structural analysis of small peptides (500-1800u). A number of different peptides, ranging in mass and amino acid sequence, are fragmented by collision with a surface in a tandem mass spectrometer and the spectra are compared with data obtained by gas-phase collisional activation. The surface-induced dissociation spectra provide ample sequence information for the peptides.

Effect of controlled local release of sodium fluoride on bone formation: filling a defect in the proximal femoral cortex.

To assess the effect of sodium fluoride (NaF) in the healing of a defect in cortical bone, an experimental model was created by the drilling of 5.0 mm holes in the proximal ends of both femora of 12 adult male New Zealand White rabbits. An interlocking intramedullary implant constructed of poly(d,l-lactic acid) containing NaF was placed in the right femur and an identical implant without NaF (sham), in the left.

Evidence for clonal anergy as a mechanism responsible for the maintenance of transplantation tolerance.

The main stimulus triggering early acute allograft rejection is known to be delivered by the allogeneic "passenger" leukocytes present within the grafts. Once these cells have been replaced by cells of recipient origin, subsequent rejection episodes are generally less frequent and less acutely destructive. How this replacement affects the cell populations responsible for allograft rejection is not known.