Medial-lateral centre of mass displacement and base of support are equally good predictors of metabolic cost in amputee walking.

Amputees are known to walk with greater metabolic cost than able-bodied individuals and establishing predictors of metabolic cost from kinematic measures, such as centre of mass (CoM) motion, during walking are important from a rehabilitative perspective, as they can provide quantifiable measures to target during gait rehabilitation in amputees. While it is known that vertical CoM motion poorly predicts metabolic cost, CoM motion in the medial-lateral (ML) and anterior-posterior directions have not been investigated in the context of gait efficiency in the amputee population. Therefore, the aims of this study were to investigate the relationship between CoM motion in all three directions of motion, base of support and walking speed, and the metabolic cost of walking in both able-bodied individuals and different levels of lower limb amputee. 37 individuals were recruited to form groups of controls, unilateral above- and below-knee, and bilateral above-knee amputees respectively. Full-body optical motion and oxygen consumption data were collected during walking at a self-selected speed. CoM position was taken as the mass-weighted average of all body segments and compared to each individual's net non-dimensional metabolic cost. Base of support and ML CoM displacement were the strongest correlates to metabolic cost and the positive correlations suggest increased ML CoM displacement or Base of support will reduce walking efficiency. Rehabilitation protocols which indirectly reduce these indicators, rather than vertical CoM displacement will likely show improvements in amputee walking efficiency.

Energy flow analysis of amputee walking shows a proximally-directed transfer of energy in intact limbs, compared to a distally-directed transfer in prosthetic limbs at push-off.

Reduced capacity and increased metabolic cost of walking occurs in amputees, despite advances in prosthetic componentry. Joint powers can quantify deficiencies in prosthetic gait, but do not reveal how energy is exchanged between limb segments. This study aimed to quantify these energy exchanges during amputee walking. Optical motion and forceplate data collected during walking at a self-selected speed for cohorts of 10 controls, 10 unilateral trans-tibial, 10 unilateral trans-femoral and 10 bilateral trans-femoral amputees were used to determine the energy exchanges between lower limb segments. At push-off, consistent thigh and shank segment powers were observed between amputee groups (1.12W/kg vs. 1.05W/kg for intact limbs and 0.97W/kg vs. 0.99W/kg for prosthetic limbs), and reduced prosthetic ankle power, particularly in trans-femoral amputees (3.12W/kg vs. 0.87W/kg). Proximally-directed energy exchange was observed in the intact limbs of amputees and controls, while prosthetic limbs displayed distally-directed energy exchanges at the knee and hip. This study used energy flow analysis to show a reversal in the direction in which energy is exchanged between prosthetic limb segments at push-off. This reversal was required to provide sufficient energy to propel the limb segments and is likely a direct result of the lack of push-off power at the prosthetic ankle, particularly in trans-femoral amputees, and leads to their increased metabolic cost of walking.

Effect of Fe(III)-ligand properties on effectiveness of modified photo-Fenton processes.

This paper examines a modified photo-Fenton (UV/Fe oxalate/H2O2) process. The degradation of oxalate in this system in the absence of Reactive Red 235 was studied using both experimentation and kinetic modelling. The degradation of Reactive Red 235 in this system was also studied. Light intensity and solution pH had large effects on the degradation of both oxalate and Reactive Red 235, with the effect of pH not due simply to speciation changes. The most important properties of the oxalate ligand in the UV/Fe oxalate/H2O2 process are that it forms Fe(III)-oxalato complexes that are easily photolysed and also is relatively unreactive with OH. radicals.

Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.

Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.

Placental arylamine N-acetyltransferase type 1: potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy.

Human arylamine N-acetyltransferase type 1 (NAT1), better known as a drug-metabolising enzyme, has been proposed to acetylate the folate catabolite p-aminobenzoylglutamate (p-abaglu) to N-acetamidobenzoylglutamate (ap-abaglu) which is a major urinary folate catabolite. Using mass spectroscopic analysis, we demonstrate the formation of ap-abaglu by recombinant human NAT1 and human placental homogenates. Using density gradient centrifugation the placental enzymic activity which acetylates p-aba and the placental enzymic activity acetylating p-abaglu both have an S(20,w) value of 3.25 S. This is the expected value for a monomer of human NAT1 (33 kDa). The specific NAT1 inhibitor 5-iodosalicylate inhibits acetylation of both p-aba and p-abaglu catalysed by either recombinant human NAT1 or placental samples as the source of enzyme. These data demonstrate that NAT1 is the major placental enzyme involved in acetylating p-abaglu.

Mechanistic insights into the inhibition of serine proteases by monocyclic lactams.

Although originally discovered as inhibitors of pencillin-binding proteins, beta-lactams have more recently found utility as serine protease inhibitors. Indeed through their ability to react irreversibly with nucleophilic serine residues they have proved extraordinarily successful as enzyme inhibitors. Consequently there has been much speculation as to the reason for the general effectiveness of beta-lactams as antibacterials or inhibitors of hydrolytic enzymes. The interaction of analogous beta- and gamma-lactams with a serine protease was investigated. Three series of gamma-lactams based upon monocyclic beta-lactam inhibitors of elastase [Firestone, R. A. et al. (1990) Tetrahedron 46, 2255-2262.] but with an extra methylene group inserted between three of the bonds in the ring were synthesized. Their interaction with porcine pancreatic elastase and their efficacy as inhibitors were evaluated through the use of kinetic, NMR, mass spectrometric, and X-ray crystallographic analyses. The first series, with the methylene group inserted between C-3 and C-4 of the beta-lactam template, were readily hydrolyzed but were inactive or very weakly active as inhibitors. The second series, with the methylene group between C-4 and the nitrogen of the beta-lactam template, were inhibitory and reacted reversibly with PPE to form acyl-enzyme complexes, which were stable with respect to hydrolysis. The third series, with the methylene group inserted between C-2 and C-3, were not hydrolyzed and were not inhibitors consistent with lack of binding to PPE. Comparison of the crystal structure of the acyl-enzyme complex formed between PPE and a second series gamma-lactam and that formed between PPE and a peptide [Wilmouth, R. C., et al. (1997) Nat. Struct. Biol. 4, 456-462.] reveals why the complexes formed with this series were resistant to hydrolysis and suggests ways in which stable acyl-enzyme complexes might be obtained from monocyclic gamma-lactam-based inhibitors.

Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of a stable acyl-enzyme complex.

beta-Lactam inhibitors of transpeptidase enzymes involved in cell wall biosynthesis remain among the most important therapeutic agents in clinical use. beta-Lactams have more recently been developed as inhibitors of serine proteases including elastase. All therapeutically useful beta-lactam inhibitors operate via mechanisms resulting in the formation of hydrolytically stable acyl-enzyme complexes. Presently, it is difficult to predict which beta-lactams will form stable acyl-enzyme complexes with serine enzymes. Further, the factors that result in the seemingly special nature of beta-lactams versus other acylating agents are unclear-if indeed they exist. Here we present the 1.6 A resolution crystal structure of a stable acyl-enzyme complex formed between porcine pancreatic elastase and a representative monocyclic beta-lactam, which forms a simple acyl-enzyme. The structure shows that the ester carbonyl is not located within the oxyanion hole and the "hydrolytic" water is displaced. Combined with additional kinetic and mass spectrometric data, the structure allows the rationalization of the low degree of hydrolytic lability observed for the beta-lactam-derived acyl-enzyme complex.

Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation.

L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the "thiol-template" proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97-100]. In the L-O-(methylserinyl)-L-valine product, no significant loss of 18O was observed. However, in the L-O-(methylserinyl)-D-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bond formation and the epimerization of the valine residue can both occur before formation of any thioester bond to the valine carboxylate in the biosynthesis of these dipeptides. The usual qualitative test for thioesterification of substrates to the synthetase, lability of enzyme-bound radiolabeled amino acid to performic acid, proved inconclusive in our hands. These results require a new mechanism for the enzymic synthesis of L-O-(methylserinyl)-L-valine and L-O-(methylserinyl)-D-valine and imply that a revised mechanism for ACV synthesis is also required.

Structure of a specific acyl-enzyme complex formed between beta-casomorphin-7 and porcine pancreatic elastase.

Mass spectrometric screening reveals that an unmodified natural heptapeptide--human beta-casomorphin-7, an internal sequence of human beta-casein that possesses opioid-like activity--reacts with porcine pancreatic elastase to form an unusually stable acyl-enzyme complex at low pH. X-ray crystallographic analysis (to 1.9 A resolution) at pH 5 shows continuous electron density linking the C-terminal isoleucine of beta-casomorphin-7 to Ser 195 through an ester bond. The structure reveals a well defined water molecule (Wat 317), equidistant between the carbon of the ester carbonyl and N epsilon 2 of His 57. Deprotonation of Wat 317 will produce a hydroxide ion positioned to attack the ester carbonyl through the favoured Bürgi-Dunitz trajectory.

Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand.

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b 38.9 A,c = 152.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 A, b = 97.6 A,c = 101.6 A, alpha = beta = gamma = 90 degrees.

An investigation of the binding of protein proteinase inhibitors to trypsin by electrospray ionization mass spectrometry.

The binding of BPTI and SBTI with trypsin has been investigated by ESI MS, using the mutant K15V-BPTI and the chemically modified RcamBPTI as controls. Although high cone voltages (+80 V) produce sharp spectra of BPTI, RcamBPTI, SBTI and trypsin alone, the complexes of BPTI, RcamBPTI and SBTI with trypsin undergo partial dissociation due to collisional activation. At lower cone voltages (+40 V) these non-covalent complexes are stable. The charge distribution on the trypsin and the inhibitors produced by gas phase dissociation of the complexes are markedly different from those of the components alone, indicating that ESI MS provides a novel probe for exploring the ionic interactions at the contact surface of proteins. Moreover, by determining the cone voltage at which the gas phase dissociation of complexes occurs it may be possible to use ESI MS to compare the binding energies of closely related complexes.

Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry.

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.

Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons.

The Link module, a 98-amino-acid domain found in hyaluronan binding proteins of human tumor necrosis factor stimulated gene 6 was overexpressed in Escherichia coli. Electrospray ionization mass spectrometry revealed that only 50% of the expressed protein had the expected wild-type molecular weight, with the remaining material having between 1 and 4 arginine to lysine substitutions, arising due to misincorporation at AGA codons. The level of misincorporation was almost completely abolished by mutation of the 4 AGA codons to CGT. This mutation to high-usage arginine codons also increased the level of heterologous protein expression. Refolding of the Link module, which occurred during the purification procedure, gave two species with different disulfide bond organizations that could be separated by high-performance liquid chromatography. One of these had a disulfide bond arrangement consistent with that found in other Link modules and, by nuclear magnetic resonance spectroscopy, was shown to be folded.

Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.

The complete amino acid sequence confirms the presence of pseudoazurin in Thiosphaera pantotropha.

The complete amino acid sequence, obtained by direct protein sequencing, of the pseudoazurin from Thiosphaera pantotropha is reported. It shows sequence identities varying from 46 to 66% with previously sequenced pseudoazurins. Previously identified conserved residues with key functions in pseudoazurins are found in the protein from T. pantotropha with the exception of glycine-39, the carbonyl group of which has been considered as a ligand to the copper, which is replaced by a serine residue. Electrospray-ionization MS (ESI-MS) has shown that pseudoazurin from T. pantotropha often contains two polypeptide species differing in molecular mass by 16 Da, presumably owing to oxidation of a methionine residue to a sulphoxide derivative. These two species have different endoproteinase Arg-C digestion patterns. Conditions for ESI-MS were identified that permitted either the retention or the loss of the single copper ion associated with the pseudoazurin. The aberrant tendency of T. pantotropha pseudoazurin to form a disulphide-bridged dimer on SDS/PAGE under some conditions is described.

Crystallization and preliminary X-ray diffraction characterisation of both a native and selenomethionyl VLA-4 binding fragment of VCAM-1.

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

Site-directed mutagenesis of beta-lactamase I: role of Glu-166.

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

beta-Lactamase ragged ends detected by electrospray mass spectrometry correlates poorly with multiple banding on isoelectric focusing.

Purified preparations of TEM-2, P99, Bacillus cereus I and B. cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry. The ES mass spectra of the B. cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends). The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends. All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends. Multiple banding patterns were seen with each beta-lactamase preparation. Although these may correlate with the presence of ragged ends in the two B. cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry. In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends. Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples. This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.