Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.

The animal and human pathogen Listeria monocytogenes secretes several virulence factors, including a phosphatidylinositol-specific phospholipase C (PI-PLC). Sufficient quantities of L. monocytogenes PI-PLC for biophysical studies were obtained by overexpression of the enzyme in Escherichia coli. The purified PI-PLC was examined in enzyme kinetics experiments using a new fluorogenic substrate, methyl-FLIP. Methyl-FLIP is a water-soluble monomeric substrate cleaved in a manner similar to the natural aggregate substrate, phosphatidylinositol (PI). Michaelis–Menten kinetics were observed with KM=61±7 μM and Vmax=120±5 μmol min−1 mg−1, corresponding to kcat=66±3 s−1. The catalysis is activated by the addition of a short-chain phospholipid, dihexanoyl phosphatidylcholine (diC6PC). The kinetics were fitted to a two-site model in which the substrate binds to the active site and diC6PC binds to a second site, with an interaction between the two sites. The result is a decrease in KM and an increase in Vmax, producing an overall four to five-fold increase in catalytic efficiency (kcat/KM). The interaction is not a regulatory mechanism, as is the case for multimeric enzymes; rather, it suggests interfacial cooperativity between the active site and a lipid-binding subsite, presumably adjacent to the active site.

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate–fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.

An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4. 10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the H-1 NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine H-delta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pK(a) of 8.0 at 6 degreesC, which is identified with the pK(a) of His32. The H-delta1 signal is modulated by rapid exchange of the H-is an element of2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 Angstrom at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor greater than or equal to 100.

The distribution of the urokinase-type plasminogen activator receptor (uPAR) on human glioma cells was examined as a function of culture conditions, using immunofluorescence and immunophotoelectron microscopy. Both uPAR colocalization with focal adhesion proteins and glioma cell motility were maximal in medium containing whole serum or a serum fraction retained by a 500,000 mol wt cutoff centrifugal concentration filter. High motility also took place in medium containing a serum fraction passed by the 500,000 cutoff filter but retained by a 100,000 cutoff filter and in minimal medium containing added vitronectin; however, under these conditions only a small percentage of the otherwise abundant focal adhesions contained colocalized uPAR. Glioma cells in minimal medium with added laminin migrated with a highly elongated morphology but without either classical focal adhesions or well-defined uPAR labeling. In contrast, glioma cells in minimal medium with no additions did not migrate, nor did they adhere well or display defined labeling patterns for focal adhesion proteins or uPAR. The results indicate that high-molecular-weight serum protein complexes promote both uPAR-focal adhesion colocalization and cell migration in glioma cells. However, conditions can be selected in which migration takes place with minimal uPAR-focal adhesion localization, as well as in the absence of apparent focal adhesions.

The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-δ 1 in a continuous assay.

Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.

In photoelectron microscopy (PEM; also known as PEEM) ultraviolet light is used to liberate electrons from the surface of a specimen through the photoelectric effect. The emitted electrons (photoelectrons) have low kinetic energies (∼1 eV) and are accelerated by an electric field between the specimen and an anode, before being imaged by a lens system. The lens system forms the image of the specimen in parallel mode, as in TEM, rather than in scanning mode, as in SEM. Image contrast arises from differences in work function and from electric or magnetic fields over the specimen surface. Micro-fields associated with surface topography are also a source of contrast. Because of the low emission energies of the electrons PEM is a highly surface-specific technique and also is relatively non-destructive. Several versions of the photoelectron microscope have been designed and constructed jointly by the University of Oregon (U0) and Portland State University (PSU), and the instrument has evolved to an advanced state of development.

The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

The three-dimensional structures of phosphatidylinositol-specific phospholipases C (PI-PLCs) from Bacillus cereus and the human pathogen Listeria monocytogenes were determined by X-ray crystallography, both in free form and in complex with the substrate-like inhibitor myo-inositol. Both enzymes share a very similar distorted (βα)8-barrel fold despite a moderate overall sequence identity of 24%. A high structural conservation is found for the active site where myo-inositol is recognized in a stereospecific fashion. Two histidine residues that are also conserved between prokaryotic and eukaryotic PI-PLCs act as a general base and a general acid during catalysis, while an arginine residue provides the electrostatic stabilization of the transition state. Based on the present crystal structures and sequence alignments, it is suggested that all Ca2+-independent PI-PLCs known so far adopt the same fold and catalytic mechanism.

: The performance characteristics of electron microscopes and probe-forming instruments depend ultimately on the focal properties and aberrations of electron lenses. A practical method of experimentally determining the properties of electron lenses is described. The method utilizes shadows cast by two meshes inserted separately in front of the lens and behind the lens to study the properties of the image of a point source. The image properties are then used to calculate the lens properties. The paraxial values of the focal length and focal distance as well as their spherical and chromatic aberrations are determined. Experimental data and the analysis are presented in the form of a tutorial that has been tested in the classroom. Discussions of the relationship between image properties and lens properties, in particular, focal point aberrations and focal length aberrations, and the various ways aberration coefficients can be defined, are included to clarify concepts in optics that are important for microscopists.

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol‐specific phospholipase C (PI‐PLC). In this report, we present the first study of the p K a values of histidines of a PI‐PLC. All six histidines of Bacillus cereus PI‐PLC were studied by 2D NMR spectroscopy and site‐directed mutagenesis. The protein was selectively labeled with 13 C ϵ1 ‐histidine. A series of 1 H‐ 13 C HSQC NMR spectra were acquired over a pH range of 4.0‐9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1 H‐ 13 C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild‐type activity. The active site histidines, His32 and His82, display p K a values of 7.6 and 6.9, respectively. His92 and His227 exhibit p K a values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The p K a value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high p K a .

Abstract: All lenses, whether for light or for electrons, have aberrations that limit their performance. In light optics the invention of the achromat over 200 years ago solved the problem of correcting spherical and chromatic aberrations; however, correcting aberrations of electron lenses remains a challenge. Spherical and chromatic aberrations constitute a fundamental barrier to improving resolution and reducing probe size in various types of electron-optical instruments. Recently, efforts have intensified to reduce probe size in microanalysis and lithography, and to improve resolution in the new emission microscopes being built for synchrotron light sources. The experiments described in this report show how an electron mirror can achieve simultaneous correction of spherical and chromatic aberrations.