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Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ETAreceptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.

The steroidogenic acute regulatory protein (StAR) helps with movement of cholesterol from the outer to the inner mitochondrial membrane in gonadal and adrenal cells thereby bringing on steroid biosynthesis. The protease-resistant domain of N-218 MLN64 and the corresponding domain of StAR, it may be inferred, likely have direct roles in moving cholesterol across the mitochondrial membrane. Decreasing pH diminishes structure, bringing on aggregation. MLN64 never goes into the mitochondria, so the protease-resistant domain of MLN64 cannot be a mitochondrial pause-transfer sequence, as was proposed for StAR.

Findings of a study show loss of estrogen receptor (ER) DNA-binding function in extracts from some primary breast tumors and in ER or ER-DNA-binding domain (DBD) exposed to thiol-reacting oxidants results from an asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. ER-DBD has several strategically located methionine resides, but they are less susceptible to oxidation than the thiol groups, and offer no protection against cysteine oxidation and so consequent loss of ER DNA-binding function.

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.

A nanoliter-chemistry station combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed to characterize proteins at the attomole level. Chemical reactions including protein digestion were carried out in nanoliter or subnanoliter volumes, followed by microspot sample deposition of the digest to a MALDI-TOF mass spectrometer. Accurate mass determination of the peptides from the enzyme digest, in conjunction with protein database searching, allowed the identification of the proteins in the protein database. This method is particularly useful for handling small-volume samples such as in single-cell analysis. The high sensitivity and specificity of this method were demonstrated by peptide mapping and identifying hemoglobin variants of sickle cell disease from a single red blood cell. The approach of combining nanoliter chemistry with highly sensitive mass spectrometric analysis should find general use in characterizing proteins from biological systems where only a limited amount of material is available for interrogation.

A strength of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is its ability to analyze mixtures without separation. MALDI mass spectrometers capable of providing a linear mass calibration over a broad mass range should find wide use in these applications. This work addresses issues pertinent to mass measurement accuracy of a time-lag focusing MALDI time-of-flight instrument and presents a new approach to improving mass accuracy by using a functional wave extraction pulse, instead of a square wave, for time-lag focusing. A model is described of an ideal extraction pulse shape that provides constant total kinetic energy for all ions. If total kinetic energy is constant, then there is an exact linear correlation between ion mass and flight time raised to the second power. Using a descending wave extraction pulse, it is demonstrated that mass accuracy of better than 30 ppm using two internal calibrants and better than 70 ppm using external calibrants can be obtained over a 25 ku mass range. The practical aspects of an instrument needed to obtain consistent mass accuracy is discussed. It is found that ion flight time shows a small dependence upon laser flux; flight times increase slightly as the flux increases. But this dependence is much smaller than is observed in continuous-extraction MALDI.

Matrix-assisted laser desorption ionization mass spectrometry has been developed for direct mass analysis of enzymatic reaction products of oligosaccharides in human blood serum without the use of extraction or chromatographic separation. Molecular labeling of the substrate is used to achieve beth the detection sensitivity and selectivity required in rapid analysis of reaction products in serum. It is found that tetramethylrhodamine (TMR)-labeled oligosaccharides provide 100-fold sensitivity enhancement over the corresponding underivatized oligosaccharides. In order to selectively retain the TMR-labeled molecules on the sample probe while washing away contaminants in a serum sample, a sample/matrix preparation method is developed. This technique provides detection sensitivity of labeled oligosaccharides in the range of hundreds of femtomoles per microliter. The mass measurement accuracy is better than 0.01% when a linear time-of-flight mass spectrometer is used. The application of the technique is illustrated for the subpicomole detection and quantitation of the conversion of the disaccharlde [Alpha]Fuc(1 [yields] 2)[Beta]Gal-TMR to the blood group B active trisaccharide [Alpha]Fuc(1 [yields] 2)[[Alpha]GaI(1 [yields] 3)][Beta]Gal-TMR, catalyzed by the blood group B galactosyltransferase present in human serum.

A time-lag focusing method is developed for the improvement of mass resolution in a linear time-of-flight mass spectrometer for matrix-assisted laser desorption/ionization (MALDI). In this technique, the ions generated by the MALDI process are extracted by a pulsed voltage. A short time delay (280 ns) is inserted in between the laser desorption/ionization event and the ion extraction. The region between the repeller and extraction grid is field-free during the delay. The time-lag extraction allows the ions generated in the region between the repeller and the extraction grid to separate according to their velocity (energy). Application, to the repeller, of the appropriate pulse voltage provides the energy correction necessary to simultaneously detect all ions of the same mass/charge regardless of their initial energy, resulting in improved mass resolution. It is demonstrated that mass resolution in the range of 3000-6000 fwhm can be obtained. With this mass resolution, isotopically resolved mass spectra are observed for peptides with masses up to 3000 Da. For proteins, such as bovine insulin, cytochrome c, and apomyoglobin, resolution in the range of 800-1000 fwhm is observed with a mass measurement accuracy better than 0.01%.

Chemically synthesize of two diastereomeric tricyclci peptides, neopetrosiamides A and B from the marine sponge Neopetrosia sp. that have ability to inhibit tumor cell invasion associated with metastasis is reported. The chemical synthesis method which used solid-phase peptide synthesis and sequential stepwise disulfide bond formation in solution could also be used to generate analogues containing methionine or norleucine instead of the methionine sulfoxide at position 24.

Studies about the steroidogenic acute regulatory protein (StAR), a mitochondrial matrix protein which acts on the outer mitochondrial membrane (OMM) to facilitate the movement of cholesterol from the outer to inner mitochondrial membrane via an unknown mechanism is reported. The findings from the study demonstrated a mechanism in which StAR is stabilized at the OMM by cholesterol to initiate its massive import into mitochondria.

The significant role of hydrophobic core for biological activity of proteins with START (steroidogenic acute regulatory protein (StAR)-related lipid transport) domains for the movement of cholesterol from the outer to inner mitochondrial membrane is demonstrated. The results obtained provide insight into the flexible form adopted by wild-type StAR due to the accommodation of more water molecules and the generation of mutant StAR generated by an alternate folding pathway.

The activity and cholesterol-binding behavior of the steroidogenic acute regulatory protein (StAR) 1 and StARD3-D7 are assessed, indicating that StARD6 has activity equal to StARD1, whereas StARD, D5, and D7 has little or no activity with adrenal mitochondria in vitro. StARD6 is expressed only in male germ-line cells and it has displayed biological and biophysical properties that have implied a role in steroidogenesis.

Multidimensional NMR studies on subtilosin A, a bacteriocin from Bacillus subtilis, are carried out to determine the complete primary and three-dimensional solution structure of peptide. Results depicted a cyclized peptide backbone with three cross-links formed between the sulfurs of Cys13, Cys 7, and Cys4 and the alpha-positions of Phe22, Thr28 and Phe31 that are showing L form of stereochemistry.