bluuebunny/crossref_metadata_2025 huggingface dataset:
The Characterizing of Particles by the Manner in which they break
Authors: Barnard W. John , Bull Frederick A.
Published: May 2005

A modified drop‐shatter test apparatus was used to study the primary breakage of particles of two solid fuels. In this apparatus many particles could be subjected to just one impact under known conditions.

About 100000 particles of each fuel, sorted by size and shape into eight separate groups, were dropped from four different heights and the fragments thus formed were sifted to determine their size distributions, that is the experimental breakage functions.

The interior of particles, of regular or irregular shape, fails under tension and the resulting fragmentation is due to stress‐activated volume flaws which give rise to coarse fragments whose distribution can be represented by a theoretical breakage function of the form

where z is the relative size of the fragments and γ is the number of stress‐activated volume flaws per particle.

The average values of γ, given by μγ = λ/(1 – e−λ), ranged from about one to five, corresponding to the formation of two to six coarse fragments per broken particle.

The probability of breakage was log‐normally related to μγ.
Fractal Description of the Structure of Fresh and Eroded Aluminum shot fineparticles
Authors: Kaye Brian H. , Leblanc John E. , Abbot Paul
Published: May 2005

Fractal dimensions are numbers between the classical whole number dimensions which can be used to describe the ruggedness of a fineparticle boundary. This communication explores the utility of fractal dimensions between 1 and 2 for characterizing the ruggedness of fineparticle profiles. This type of fractal boundary is evaluated by estimating the perimeter of a profile of a fineparticle at various levels of scrutiny and then plotting a graph of the logarithm of the perimeter estimates against the logarithm of a unit representing the scale of scrutiny. Such a graph is known as a Richardson plot. Boundaries describable by fractal dimensions exhibit characteristic datalines on this type of graph.

Aluminum shot fineparticles produced by the break up of turbulent jet of molten metal have irregular shapes because of the fluid turbulence in the regions where they solidify, but a smooth texture because of the effect of surface tension forces as the fineparticle solidifies. It is shown in this communication that the dataline on a Richardson plot of the estimated perimeter of an aluminum shot fineparticle consists of two regions. The first portion, at coarse levels of scrutiny, permits the calculation of a fractal dimension descriptor of the ruggedness of the profile. A second dataline at high resolution is parallel to the scrutiny axis indicating that at such levels of inspection the profile is effectively smooth and euclidean. Studies are reported of the changes in the ruggedness of an eroding aluminum shot fineparticle suspended in acid. It is shown that fractals can be used to describe the progress of corrosion and that, as corrosion proceeds, the euclidean portion of the Richardson plot of perimeter estimate recedes and disappears within the levels of scrutiny employed in these experiments.
Mercury Porosimetry Studies III: A Check on the Validity of the Critical pressure technique for porous powders
Authors: Davidson John A.
Published: May 2005

In any determination of the internal porosity (intra particle porosity) by mercury porosimetry, the penetration curve must be interpreted in such a way that the contribution of the interparticle porosity is deleted. In an earlier communication an experimental technique, known as the critical pressure technique, was developed to accomplish this goal. In this communication a hot stage microscopy image analysis technique is described for the determination of the intra particle porosity of PVC resins. This enables the validity of the critical pressure method to be independently tested. On 12 samples of PVC resin a 1 – 1 correlation between the two methods was found indicating the validity of the critical pressure technique. Another porosity determination technique described earlier, based on the uptake of wetting oil, is also discussed in the light of these findings. This work also illustrates that hot stage microscopy, combined with image analysis, can be used to determine the glass transition of polymers on samples as small as 1·10−8 grams.
Measurement of velocities and diameters of small particles by using fast digital circuits for the on‐line evaluation of laser‐doppler‐signals
Authors: Bauckhage Klaus , Schöne Armin
Published: May 2005

Inspection of the scattered light by two photodetectors makes it possible to investigate the velocities of droplets as well as their size in disperse systems and has some advantages against related methods. When this method is used, the size of the droplets can be derived from the phase difference of the signals from the first and the second photodetector.

Special problems of this method are discussed in the paper. A system with fast signal processing facilities, working mostly digitally, was developed in order to solve these problems. This system derives several digital signals from the signals of high frequencies that are gained from the photodetectors. These digital signals are combined by digital circuits of the ECL‐type. The resulting pulse series are fed into several digital counters. The data from the counters and from some other inputs are further processed by a real time computer that was developed for this special task, using an 8088 micro processor. This real time computer is connected to an ordinary personal computer by a parallel interface.
Particle Deposition in High Capacity Preseparators
Authors: Fissan Heinz , Opiolka Siegfried
Published: May 2005

High capacity preseparators are used to reduce bounce and blow‐off of large particles in cascade impactors. In this paper, results are presented of a detailed analysis of the deposition of particles in two slightly different preseparators of the impactor‐type which are used in connection with the Andersen Stack Sampler. The advantage of using any preseparator for aerosols mixed with large particles (x 10 μm) is clearly shown.

A preseparator which is integrated into the impactor has the advantages of smaller overall size, accessible surfaces and, therefore, easier handling. The results of the study show that the reduction of unwanted particle losses is rather small when compared with a separate preseparator.
The Storage and Handling of Polymer Latices Used as Particle Standards
Authors: Wilkinson Michael , Hearn John , Karpowicz Franciszek , Chainey Malcolm
Published: May 2005

Latices of 2, 4 and 9 μm diameter intended for use a particle standard have been examined under a variety of storage and handling conditions. The effects of light exposure, temperature variation and storage in isotonic saline were studied as well as the handling conditions, such as multiple sampling, dispersion technique and effects of foaming. Particle sizes and particle number densities were remarkably constant, even under the most extreme conditions investigated.
An Optical Particle Counting System for Testing Industrial Pulse‐Cleaned Cartridge Dust Collectors
Authors: Liu Benjamin Y. H. , Pui David Y. H. , Schaller Robin E. , McDonald Bruce N. , Johnson Todd W.
Published: May 2005

An optical particle counting system has been developed for testing industrial pulse‐cleaned cartridge dust collectors. The system is applied to measure fractional penetration of a full scale dust collector and to study its short term and long term dynamic behavior. Results show collector efficiencies for dust loadings of 23 g/m3 exceeding 99.999% over the particle size range from 0.5 μm to 10 μm, with a minimum fractional penetration occurring at approximately 2 μm.
Vision‐Guided Flame Control Using Fuzzy Logic and Neural Networks
Authors: Tao Wenjing , Burkhardt Hans
Published: May 2005

This paper presents an application of fuzzy and neural network techniques to a vision‐guided closed loop control for stationary luminous flames. The image processing technique is used to analyze and identify the process states. Fuzzy control strategy avoids the difficulty in establishing a mathematical model for an ill‐defined process. Expert knowledge and training patterns can be incorporated into fuzzy rules, which are represented in the form of neurons. The use of a neural network makes it easy to increase the number of control parameters and provides the system the possibility to adjust its performance automatically.
Extinction of Radiation in Sterically Interacting Systems of Monodisperse Spheres. Part 2: Experimental results
Authors: Kräuter Udo , Riebel Ulrich
Published: May 2005

The Bouguer‐Lambert‐Beer law (BLBL), commonly used to describe the extinction of radiation in disperse systems, is known to be valid in the limit of low concentrations only. In Part [1] it was shown that steric particle‐particle interactions lead to important effects in the range of high particle concentration and two versions of a more general extinction equation including steric interactions were derived for the regimes of fraunhofer diffraction and ray propagation in this part experimental evidence for the effects of steric interactions on the extinction of radiation in suspensions of monodisperse spherical particles is given. Various experiments on light extinction and on ultrasonic extinction are combined to cover a broad range of particle size parameters, mechanisms of particle‐wave interaction and receiver apertures.
Dry Mechanical Dispersion of Submicron Particles
Authors: Leschonski Kurt , Benker Bernd , Bauer Ulrich
Published: May 2005

Concerning the use of air classifiers with cut sizes in the micron range and lower, it is necessary to disperse the feed material properly in an air flow to avoid agglomerates larger than the desired cut size. Commonly used dispersing devices such as injectors use large amounts of air. To reduce the size and costs of the subsequent air classifiers, two new types of dispersing systems were developed, one design being related to a brush feeder and the other to a pin mill. Both apply mechanical rather than fluid mechanical forces in the dispersion process. The brush disperser achieves the same size distributions as the pin mill disperser with much less machinery. Its properties as a feeding and dispersing system are shown and its ability to disperse particles in the submicron range is confirmed by two independent particle size analysis systems: a new diffraction spectrometer and an impactor.
On Measuring and Modeling Local Average Particle Velocities for particle ensembles in classifying systems
Authors: Riehle Claus
Published: May 2005

In most multi‐phase flow problems, the particulate phase is exposed to an external field which causes dispersion. Therefore, local velocity measurements of the disperse phase are no longer equivalent with respect to averaging in time and averaging in volume. While the local time‐averaged velocity still characterizes the transport of the ensemble in the Eulerian sense, one has to be be careful in modeling this velocity average by considering the ensemble's composition. It is shown for different particle ensembles that the conventional particle velocity average M1,0 calculated with respect to the dispersion relationship and a particle size number density distribution is far below the measured ensemble average; the deviation depends on the width of the particle size distribution. It is deduced that Eulerian particle velocity values referring to a certain time interval can be modeled by a ratio of velocity moments M2,0/M1,0 calculated with particle size number distributions referring to a certain probe volume. This relationship was confirmed by measurements.
Particle Size Determination by Laser Reflection: Methodology and problems
Authors: Monnier Olivier , Klein Jean‐Paul , Ratsimba Berthe , Hoff Christian
Published: May 2005

The particle size distribution of crystalline solids has progressively become a key parameter in manufacturing processes, as important as chemical purity. Among the particle size determination and counting systems available on the market, very few offer the possibility of continuous in situ monitoring of the particle size evolution during crystallization. For this reason, much interest has been aroused by the appearance of the Par Tec 100, patented by Laser Sensor Technology [1, 2]. A study has been carried out in a stirred vessel to verify the precision and reproducibility of particle size measurement and elucidate the influence of experimental parameters on data accessible with this instrument. Optimum reproducibility has logically been achieved by fixing the highest possible cycle time and taking the mean of several cycles. Determinations with the Par Tec 100 are influenced variously, according to whether they relate to the total number of particles counted or to the mean size. Thus, the number of counts measured by a particle size probe largely depends on the operating conditions and more particularly on the hydrodynamic conditions, solvent, temperature and focal point position. Its dependence relative to the concentration of the solid in suspension is normal and linear for a solid and for a given monodisperse sample. To establish the relationship between the number of counts and the population density would therefore necessitate delicate calibration on a case‐by‐case basis. The mean size determined does not depend on suspension homogeneity, provided that the stirring speed is sufficient for a statistically significant total count. On the other hand, for a given sample, a displacement of the focal point can lead to considerable variations in the size determined. The optimal focal point position for small sizes is in fact highly sensitive. Lastly, the optimal position of the focal point is considerably dependent on the true size of the particles, which means that this counter is unsuitable for the precise analysis of a dispersed sample since each particle size class would require a different setting of the focal point. In addition, the sizes determined, irrespective of the products studied, appear to be underestimated for large particles and over estimated for small particles.
Experimental Studies of Electrically Active Fibrous Filter Loading
Authors: Walsh Declan , Stenhouse Ian
Published: May 2005

Electrically active fibrous filters, that is fibrous filters whose fibres carry a permanent electric charge, are an increasingly popular alternative to conventional fibrous filters in applications where low pressure drop and high collection efficiencies are critical. The advantage of these materials is the additional collection efficiency, due to electrostatic mechanisms, that can be achieved without pressure drop increase. The efficiency of these materials can fall as they are loaded with aerosol particles, so it is necessary that proper account be taken of this process during use. A complete understanding of the mechanisms responsible for this reduction in efficiency has not yet been reached. An experimental study has been undertaken at Loughborough University of Technology investigating the loading behaviour of a mixed fibre type electrically active material. The experimental techniques used in this work and experimental findings with a range of aerosol particle sizes are reported here.
Phase‐Doppler Anemometry with the Dual Burst Technique for measurement of refractive index and absorption coefficient simultaneously with size and velocity
Authors: Onofri Fabrice , Girasole Thierry , Gréhan Gérard , Gouesbet Gérard , Brenn Günter , Domnick Joachim , Xu Tian‐Hua , Tropea Cameron
Published: May 2005

The principle of the dual burst technique (DBT) based on phase‐Doppler anemometry (PDA) is proposed for simultaneous particle refractive index, size and velocity measurements. This technique used the trajectory effects in PDA systems to separate the two contributions of the different scattering processes. In the case of forward scattering and refracting particles, it is shown that from the phase of the reflected contribution, the particle diameter can be deduced, whereas from the refracted contribution the particle refractive index and velocity can be obtained. Furthermore, the intensity ratio of these two scattering processes can be used for absorption measurements. Simulations based on generalized Lorenz‐Mie theory and experimental tests using monodispersed droplets of different refractive indices and absorption coefficients have validated this technique.
Simultaneous Measurement of Velocity and Equivalent Diameter of Particles with arbitrary shape
Authors: Terhaag Ulrich , Renz Ulrich
Published: May 2005

Only a few optical techniques are available to measure simultaneously the velocity and size of coarse particles dispersed in a fluid. Since particles are randomly shaped with a rough surface and inhomogeneous with respect to the refractive index, phase‐based sizing techniques are not reliable. The applied optical sizing technique is based on light intensity measurement of a scattering particle crossing a top‐hat laser beam. This optical set‐up was first described by Gréhan et al. In the range of geometrical optics, the sampled light at the receiving system is proportional to the square of the equivalent particle diameter within the diameter range 20–700 μm

Velocity information is obtained by using a smaller concentric laser Doppler probe volume. Coincidence of the signals is guaranteed by the optical set‐up, since the smaller probe volume diameter defines the triggering event. To demonstrate the performance of the system, some diameter distributions measured with the top‐hat beam system are compared with results for a commercial laser diffraction system for different materials. The system is applied to obtain size‐correlated velocity information of a particle‐laden two‐phase flow at a single‐sided backward‐facing step.
On the measurements of particles by imaging methods: Theoretical and Experimental Aspects
Authors: Ren Kuan Fang , Gouesbet Gérard , Géhan Gérad , Lebrun Denis , Özkul Cafer , Kleitz Alain
Published: May 2005

Imaging techniques in particle sizing have always been important. Nevertheless, owing to the progress in CCD cameras, these techniques have found new potential which strongly depends on the capability to describe the image formation. Based on a combination of the generalized Lorenz‐Mie theory and the diffraction theory, the image characteristics are described, taking into account the beam size, particle location, direction of observation and collecting optics characteristics. In the case of forward detection, a finite coherence is introduced and the results are compared with those obtained by a convolution approach, with the point spread function assumed to be Gaussian. Then some experimental results are displayed which underline the improvement achieved in particle size measurement.
Image‐Sedimentation technique for practical size analysis
Authors: Ma Xinghua , Wan Mingrui
Published: May 2005

A method called “image‐sedimentation”, based on the application of Stokes' law to suspension and image techniques, is proposed for the rapid analysis of particle size distributions. A parallel beam of optical light, after passing through a cell containing particles, is measured with a linear image sensor with 2048 photodetectors to determine the attenuated light intensity displayed on a monitor screen. The settling height between any given point and the liquid surface can be measured accurately by use of this image technique without any additional mechanical device to move either the light beam or the glass cell containing the suspension. Consequently, the total time required for the determination of the particle size distribution, typically of a carborundum powder having a median diameter of 5 μm, takes only 5 min as compared with more than 20 min by using other instruments based on the sedimentation principle.
A modified high‐output, size‐selective aerosol generator
Authors: Chein Hungmin , Chou Charles C.‐K.
Published: May 2005

A compact and commercial aerosol generator capable of generating narrowly size‐distributed aerosols with high mass concentrations was designed, fabricated and tested. The aerosol generator, consisting of a Delavan simplex nozzle (Model 30610‐4), an L‐shaped settling chamber and a virtual impactor with a clean air core, was modified and improved from Chein and Lundgren's work [20] to be more compact and readily commercial. The performance of the aerosol generator was evaluated using corn‐oil, sodium chloride and uranine solutions. The results indicated that the cornoil droplets produced by the generator had a mass medium aerodynamic diameter (MMAD) of 7.20 ± 0.32 μm with a geometric standard deviation (GSD) of 1.48 ± 0.01 and the aerosol generation rate was 13.8 ± 1.3 mg/min. Solid aerosols generated from NaCl solution were found to have an MMAD in the range 1.39–4.88 μm with a GSD of 1.34–1.47 with the volumetric solution concentration varying from 0.1% to 9%. At the same time, the aerosol generation rate varied from 0.27 ± 0.05 to 15.8 ± 1.8 mg/min. depending on the solution concentration and the particle size produced. In addition, a 0.01% uraniane solution was tested to generate a submicron aerosol with an MMAD of 0.93 μm and a GSD of 1.48.
pH Dependence of 2,3‐diphosphoglycerate binding to human hemoglobin Ao at 21.5°C
Authors: Hobish Mitchell K. , Powers Dennis A.
Published: May 2005

Rate equilibrium dialysis was used to measure the binding of 2,3‐diphosphoglycerate (DPG) to human oxy‐ and deoxyhemoglobin AO over the range pH 5–9, at 21.5°C. This approach yielded an accurate, precise, and self‐consistent set of model‐independent association constants. These data were successfully fitted to a thermodynamic model which is fuctionally similar to a Hill equation. The isotherms generated by this fitting procedure appear to intersect at low pH and converge at high pH. This apparent convergence at high pH is consistent with results obtained by oxygen equilibria studies performed under conditions of saturating DPG. These calculated isotherms were used to determine the enhancement of the Bohr effect as a function of pH. These results are consistent with data obtained by pH stat measurements by other investigators.

This paper presents the first in a series of studies that will provide a systematic characterization of the interaction between hemoglobin and DPG.
Kinetic characterization of early intermediates in the folding of E. coli tryptophan‐synthase β2 subunit
Authors: Blond Sylvie , Goldberg Michel E.
Published: May 2005

This report describes the use of fluorescence energy transfer between an intrinsic energy donor (tryptophan 177) and two chemically added acceptors to study intermediates in the folding of the β2 subunit of E. coli tryptophan‐synthase. Two early folding steps are thus identified and characterized. One is very rapid (its rate constant at 12°C is 0.02 sec−1) and corresponds to the folding of the N‐terminal domain into a structure whose overall features approximate well those of the native domain. The second step is somewhat slower (its rate constant at 12°C is 0.008 sec−1) and involves a conformational rearrangement of the N‐terminal domain brought about by the interactions between the N‐and C‐terminal domains within a monomeric β chain. This brings to five the number of intermediates which have been identified and ordered on the folding pathway of the dimeric β2 subunit.
Proteolytic dissection of a hapten binding site
Authors: Sen Jyoti , Beychok Sherman
Published: May 2005

IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten.
Affinity of nonhomologous amphiphilic peptides toward a monoclonal antibody raised against apolipoprotein A‐I
Authors: Khalil Adli , Bramson Neal , Kezdy Ferenc J. , Kaiser Emil Thomas , Scanu Angelo M.
Published: May 2005

Monoclonal antibodies against human apolipoprotein A‐I (apoA‐I) were generated by the hybridoma technique. Clone G‐10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme‐linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA‐I bound to the surface of the microtiter plate, apoA‐I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA‐I/anti‐apoA‐I‐complex, Kd = 2 × 10−9 M, was significantly lower than that of the complex resulting from the interaction between anti‐apoA‐I and either apoA‐I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G‐10 recognizes in antigenic peptides an α‐helical secondary structure of defined hydrophilic‐lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.
Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi
Authors: Warren R. A. J. , Beck C. F. , Gilkes N. R. , Kilburn D. G. , Langsford M. L. , Miller R. C. , O'Neill G. P. , Scheufens M. , Wong W. K. R.
Published: May 2005

Cellulomonas fimi produces an endoglucanase and an exoglucanase which bind strongly to cellulose. Each enzyme contains three distinct regions: a short sequence of about 20 amino acids containing only proline and threonine (the ProThr box); an irregular region, rich in hydroxyamino acids, of low charge density, and which is predicted to have little secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure. The Pro‐Thr box is conserved almost perfectly in the two enzymes. The irregular regions are 50% conserved, and the conserved sequences include four Asn‐Xaa‐Ser/Thr sites. The ordered regions appear not to be conserved, but the potential active sites both have the sequence Glu‐Xaa7‐Asn‐Xaa6‐Thr; they occur at widely separated sites in the two regions. The order of the regions is reversed in the two enzymes: irregular‐Pro‐Thr box‐ordered in the endoglucanase; ordered‐Pro‐Thr box irregular in the exoglucanase. The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.
Ion pairs in alpha helices
Authors: Sundaralingam M. , Sekharudu Y.C. , Yathindra N. , Ravichandran V.
Published: May 2005

A survey of 47 globular proteins was made to determine the probability of occurrence of ion pairs separated by 1, 2, 3, … and 8 residues in the alpha helices. As a control, the probability of occurrence of like charged pairs was also determined. The survey showed that ion pairs of the type i, i±3 and i, i±4 are the most predominant. Such a preference was not observed for like charged pairs. The observed frequency of ion pairs is significantly greater than their expected frequency. The normalized frequencies of occurrence of the ion pairs were also found to increase generally with the helix length. These results indicate that the ion pairs may contribute to the stability of solvent‐exposed alpha helices. Since the stabilization of protein secondary structure, these results may throw light on the mechanism of protein folding.
Three‐dimensional structure of neuraminidase of subtype N9 from an avian influenza virus
Authors: Baker A.T. , Varghese J.N. , Laver W.G. , Air G.M. , Colman P.M.
Published: May 2005

Neuraminidases from different subtypes of influenza virus are characterized by the absence of serlogical cross‐reactivity and an amino acid sequence homology of approximately 50%. The three‐dimensional structure of the neuraminidase antigen of subtype N9 from an avian influenza virus (A/tern/Australia/G70c/75) has been determined by X‐ray crystallography and shown to be folded similarly to neuraminidase of subtype N2 isolated from a human influenza virus. This result demontrates that absence of immunological cross‐reactivity is no measure of dissimilarity of polypeptide chain folding. Small differences in the way in which the subunits are organized around the molecular fourfold axis are observed. Insertions and deletions with respect to subtype N2 neuraminidase occur in four regions, only one of which is located within the major antigenic determinants around the enzyme active site.
Modeling the biochemical differences between rabbit muscle and human liver phosphorylase
Authors: Rath Virginia L. , Newgard Christopher B. , Sprang Stephen R. , Goldsmith Elizabeth J. , Fletterick Robert J.
Published: May 2005

Glycogen phosphorylases catalzye the regulated breakdown of glycogen to glucose‐1‐phosphate. In mammals, glycogen phosphorylase occurs in three different isozymes called liver, muscle and brain after the tissues in which they are prefer entially expressed. The muscle isozyme binds and is activated cooperatively by AMP. In contrast, the liver enzyme binds AMP noncooperatively and is poorly activated. The amino acid sequence of human liver phosphorylase is 80% identical with rabbit muscle phosphorylase, and those residues which contact AMP are conserved. Using computer graphics software, we replaced side chains of the known rabbit muscle structure with those of human liver phosphorylase and interpreted the effects of these changes in order to account for the biochemical differences between them. We have identified two substitutions in liver phosphorylase potentially important in altering the cooperative binding and activation of this isozyme by AMP.
Pattern descriptors and the unidentified reading frame 6 human mtDNA dinucleotide‐binding site
Authors: Webster Teresa A. , Lathrop Richard H. , Smith Temple F.
Published: May 2005

In an effort to identify the structural elements essential to a given protein function a new pattern‐directed inference system has been developed. It has been employed to identify a potential dinucleotide‐binding domain within the human mitochondrial unidentified reading frame 6 product, thereby supporting an earlier study that this gene may encode a NADH dehydrogenase subunit.
Site‐directed mutations altering methyl‐accepting residues of a sensory transducer protein
Authors: Nowlin Dawn M. , Bollinger John , Hazelbauer Gerald L.
Published: May 2005

The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methylaccepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide‐directed, site‐specific mutagenesis to substitute an alanyl residue at each of the five methyl‐accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl‐accepting activity of other sites, including one case of activation of a site not methylated in wild‐type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl‐accepting sites on the transducer lengthened the time required to reach an adapted state.
Unusual zymogen‐processing properties of a mutated form of prochymosin
Authors: McCaman Michael T. , Cummings Diana Begley
Published: May 2005

Site‐specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N‐terminal propeptide had been deleted and an eighth residue had been substituted. This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH. Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser‐Lys bond in the prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe‐Leu bond, but at a rate severalfold slower than the authentic zymogen. These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid‐catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.
Conformation of alamethicin in phospholipid vesicles: Implications for insertion models
Authors: Cascio M. , Wallace B. A.
Published: May 2005

The secondary structure of alamethicin, a membrane channel‐forming polypeptide, has been examined by circular dichroism spectroscopy to determine the relationship of its conformation in organic solution to its conformation in a membrane‐bound state. The spectrum of alamethicin in small unilamellar dimyristoyl phosphatidylcholine vesicles is significantly different from its spectrum in 10% methanol/acetonitrile, the solvent from which it was crystallized (Fox and Richards: Nature 300:325–330, 1982), as well as its spectrum in methanol, the solvent in which NMR studies have been done (Banerjee and Chan: Biochemistry 22:3709–3713, 1983). This suggests that structural models based on studies of the molecule in organic solvents may not be entirely appropriate for the membrane‐bound state. To distinguish between different models for channel formation and insertion, two different methods were used to associate the alamethicin with vesicles; in addition, the effect of oligomerization on the conformation of the membrane‐bound state was investigated. These studies are consistent with a modified insertion model in which alamethicin monomers, dimers, or trimers associate with the bilayer and then spontaneously oligomerize to form a prechannel with a higher helix content. This aggregate could then “open” upon application of an appropriate gating transmembrane potential.
Internal motion of a tryptophan residue in Streptomyces subtilisin inhibitor: Deuterium nuclear magnetic resonance in solution
Authors: Akasaka Kazuyuki , Inoue Tomoko , Tamura Atsuo , Watari Hiroshi , Abe Kimie , Kainosho Masatsune
Published: May 2005

Deuterium NMR spectroscopy was used to study internal motions of a deuterium‐labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one‐quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25°C, showing that it exits in an equilibrium mixture of two conformers, in one of which the typtophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the α‐lobe and the beta;‐lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN′, the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the “loose” conformer over the “tight” conformer.
Structural alignment and analysis of two distantly related proteins: Aplysia limacina myoglobin and sea lamprey globin
Authors: Pastore Annalisa , Lesk Arthur M. , Bolognesi Martino , Onesti Silvia
Published: May 2005

Two new globin structures have recently been determined at high resolution: the globin from the mollusc Aplysia limacina at 1.6 A resolution and a new refinement of the structure from sea lamprey. Two amino acid sequences of these homologous molecules have only 30% residue identity in an optimal alignment. We discuss some of the problems arising in the alignment of Aplysia globin with other globins of known structure, a challenging problem because of the distant relationship. Four independent approaches were applied to the alignment of the Aplysia and lamprey globins, including those based on individual sequence comparisons, structural analysis, and the relatively new method of templates or fingerprints derived for an entire family of proteins. We also compare these two new structures with what is already known about the globin family. A detailed description of the two structures shows that the two molecules contain the main structural features common to all the globins so far studied with several minor but interesting hitherto unobserved variations.
Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: A pentacoordinated binding of the SCN− ion to the zinc at high pH
Authors: Eriksson A. Elisabeth , Kylsten Per M. , Jones T. Alwyn , Liljas Anders
Published: May 2005

The binding of four inhibitors—mercuric ion, 3‐acetoxymercuri‐4‐aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion—to human carbonic anhydrase II (HCA II) has been studied with X‐ray crystallography.

The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 Å resolution. Mercuric ions are observed at both nitrogens in the His‐64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys‐206.

The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 Å, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closet to the zinc ion is 3.1 Å away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc.

The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 Å resolution using the least‐squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN− ion is 1.9 Å from the zinc ion but shifted 1.3 Å with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the Oγl atom of Thr‐199. This is due to the inability of the Oγl atom of Thr‐199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN− molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so‐called “deep” water molecule of the native enzyme. The zinc‐bound water molecule is 2.2 Å from the zinc ion and 2.4 Å from the SCN− nitrogen. In addition, this water is hydrogen bonded to the Oγl atom of Thr‐199 and to another water molecule.

We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3− ion are ligated to the zinc ion.
Comparison of CD spectra in the aromatic region on a series on variant proteins substituted at a unique position of tryptophan synthase α‐subunit
Authors: Ogasahara Kyoko , Sawada Shintaro , Yutani Katsuhide
Published: May 2005

CD spectra in the aromatic region of a series of the mutant α‐subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25°C. The measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild‐type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild‐type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of thewild‐type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild‐type proteins. These residues are polar. In the third group, the CDvalues of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild‐type protein, except for oneband at 276.5 nm. these results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residuesat position 49.
The 15 N‐terminal amino acids of hexokinase II are not required for in vivo function: Analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae
Authors: Ma Hong , Bloom Leslie M. , Dakin Susan E. , Walsh Christopher T. , Botstein David
Published: May 2005

The function of the N‐terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form).This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short from protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild‐type protein dimerizes. Kinetic studies show that the enzymatic activities are very similarto wild‐type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form ofthe enzyme is similar to the wild‐type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N‐terminalamino acids of hexokinase II are not required in vivo either for phosporylation of hexoses or for glucose repression.
Substrate specificities in class A β‐lactamases: Preference for penams vs. cephams. The role of residues 237
Authors: Healey William J. , Labgold Marc R. , Richards John H.
Published: May 2005

Site saturation mutagenesis has been carried out at Ala‐237 in RTEM‐1 β‐lactamase to assess the role of this site in modulating differences in specificity of β‐lactamases for penams vs. cephams as substrates. (An Ala‐237 Thr mutation had previously been shown to increase activity on cephems by about 30–80%.1,2) Screening of all 19 possibles mutants on penams and cephems revealed the even more active Ala‐237 Asn mutant. Detailed kinnetic analysis showns that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild‐type enzyme. Both mutations reduce the activity toward penams to about 10% that of RETM‐1 β‐lactamase and lower by about 5°C the tempreature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most intresting aspect of these results is that two quite disparate amino acids, theronine and asparagine, when intorduced for Ala‐237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.
Deletions and replacements of omega loops in yeast iso‐1‐cytochrome c
Authors: Fetrow Jacquelyn S. , Cardillo Thomas S. , Sherman Fred
Published: May 2005

Ω(Omega)‐loops are protein secondary structural elements having small distance between segment termini. It should be possible to delete or replace certain of these Ω‐loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso‐1‐cytochrome c from the yeast Saccharomyces cerevisiae were in investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different Ω‐loops were individually deleted, or in which one Ω‐loop was replaced with five different segments. Deletion encompassing amino acid positions 27–33 and79–83 either prevented synthesis of the holoprotein, or produced highly labile iso‐1‐cytochromes c, whereas deletions encompassing position 42–45 and 48–55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue position 24–33) with the same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso‐1‐cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.