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At least 127 records · Page 7Linked to original sources
PubMed1993

Copper thick film sintering studies in an environmental scanning electron microscope.

The significance of the ElectroScan environmental scanning electron microscope (ESEM) as a processing tool for studying dynamic morphological changes under controlled temperature/atmosphere conditions was evaluated. The ability to observe dynamic processes in situ, which cannot be achieved by other means, is critical to understanding microstructural formation. Processing of printed copper thick films on ceramics was used as a test case, wherein morphological changes associated with the steps of organic binder removal and sintering of copper particles were observed/examined in real time. Good agreement was seen between microstructures obtained in the ESM and those achieved in a belt furnace when similar process variables were used. When processed in atmospheres which were proven to induce sintering in a conventional belt furnace, sintering was evident in both cases, and the microstructural changes were documented on video-tapes in real time. Determination of critical event temperatures was achieved--that is, binder burnout occurring between 270 degrees and 350 degrees C, onset of oxidation at 520 degrees C, and sintering starting at 770 degrees C. It was thus verified that the microstructural changes during the copper thick film sintering process can be observed in situ using an ESEM.

Conductometry↗
PubMed1996

X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.

Amino Acid Sequence↗
PubMed1994

Conductimetric assays for the hydrolase and transferase activities of phospholipase D enzymes.

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

Animals↗
PubMed1996

Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.

Amino Acid Sequence↗
PubMed1997

Measurement of phospholipase D activity.

Phosphodiesteric cleavage of phosphatidylcholine by members of a growing family of phospholipases D produces choline and phosphatidic acid. These enzymes can also catalyse a transphosphatidylation reaction in which the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. PLD enzymes are found in a variety of organisms including bacteria, yeast, plants, and vertebrates. In mammalian systems, biochemical and cell biological approaches have identified phosphatidic acid as a mediator (or progenitor of mediators) that play important roles in the transduction of extracellular signals. Phosphatidic acid or its metabolites may be regulators of key cellular processes such as the control of intracellular protein trafficking, secretion, and alterations in cell morphology and motility. This review discusses methods for the determination of PLD activity both in vitro and in intact cells.

Animals↗
PubMed2002

Nonradioactive analysis of phosphatidylinositides and other anionic phospholipids by anion-exchange high-performance liquid chromatography with suppressed conductivity detection.

Phosphatidylinositol 4,5-biphosphate (PIP(2)) modulates the function of numerous ion transporters and channels, as well as cell signaling and cytoskeletal proteins. To study PIP(2) levels of cells without radiolabeling, we have developed a new method to quantify anionic phospholipid species. Phospholipids are extracted and deacylated to glycero-head groups, which are then separated by anion-exchange HPLC and detected by suppressed conductivity measurements. The major anionic head groups can be quantified in single runs with practical detection limits of about 100 pmol, and the D3 isoforms of phosphatidylinositol phosphate (PIP) and PIP(2) are detected as shoulder peaks. In HeLa, Hek 293 and COS cells, as well as intact heart, PIP(2) amounts to 0.5 to 1.5% of total anionic phospholipid (10 to 30 micromol/liter cell water or 0.15 to 0.45 nmol/mg protein). In cell cultures, overexpression of Type I PIP5-kinase specifically increases PIP(2), whereas overexpression of Type II PI4-kinase can increase both PIP and PIP(2). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and the D3 isomers of PIP(2) are detected after treatment of cells with pervanadate; in yeast, overexpression of a phosphatidylinositol 3-kinase (VPS34) specifically increases phosphatidylinositol 3-phosphate (PI3P). Using isolated cardiac membranes, lipid kinase and lipid phosphatase activities can be monitored with the same methods. Upon addition of ATP, PIP increases while PIP(2) remains low; exogenous PIP(2) is rapidly degraded to PIP and phosphatidylinositol (PI). In summary, the HPLC methods described here can be used to probe multiple aspects of phosphatidylinositide (Ptide) metabolism without radiolabeling.

1-Phosphatidylinositol 4-Kinase↗
PubMed2000

Specific detection of membrane-toxic substances with a conductivity assay.

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds.

Benzalkonium Compounds↗
PubMed1982

Conductivometric determination of urinary oxalate with oxalate decarboxylase.

An enzymatic method for determination of urinary oxalate is described: the acidified urine samples are extracted with chloroform. This manipulation improves the blank values considerably. 1 ml of extracted urine is incubated with oxalate decarboxylase. The CO2 released from the medium is absorbed by Sr(OH)2. The change in conductivity measured in the Sr(OH)2 solution is linearly proportional to the oxalate concentration in urine and the method is specific for oxalate. The mean recovery is 93.2 +/- 2.5%. The coefficient of variation calculated from 28 determinations is 12.6%. The detection limit is 35 nmol. 1 ml of urine is usually sufficient for determination. The mean 24 h urine oxalate excretion of 11 healthy men and 16 women was 240 +/- 20 mumol.

Carboxy-Lyases↗