The human pancreatic alpha-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose.

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor.

Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain.

Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151.

The X-ray structure of human trypsin 1 has been determined in the presence of diisopropyl-phosphofluoridate by the molecular replacement method and refined at a resolution of 2.2 A to an R-factor of 18%. Crystals belong to the space group P4, with two independent molecules in the asymmetric unit packing as crystallographic tetramers. This study was performed in order to seek possible structural peculiarities of human trypsin 1, suggested by some striking differences in its biochemical behavior as compared to other trypsins of mammalian species. Its fold is, in fact, very similar to those of the bovine, rat and porcine trypsins, with root-mean-square differences in the 0.4 to 0.6 A range for all 223 C alpha positions. The most unexpected feature of the human trypsin 1 structure is in the phosphorylated state of tyrosine residue 151 in the present X-ray study. This feature was confirmed by mass spectrometry on the same inhibited sample and also on the native enzyme. This phosphorylation strengthens the outstanding clustering of highly negative or highly positive electrostatic surface potentials. The peculiar inhibitory behaviour of pancreatic secretory trypsin inhibitors of the Kazal type on this enzyme is discussed as a possible consequence of these properties. A charged surface loop has also been interpreted as an epitope site recognised by a monoclonal antibody specific to human trypsin 1.

Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.

The matrix protein VP40 of Ebola virus is believed to play a central role in viral assembly as it targets the plasma membrane of infected cells and subsequently forms a tightly packed layer on the inner side of the viral envelope. Expression of VP40 in Escherichia coli and subsequent proteolysis yielded two structural variants differing by a C-terminal truncation 114 amino acid residues long. As indicated by chemical cross-linking studies and electron microscopy, the larger polypeptide was present in a monomeric form, whereas the truncated one formed hexamers. When analyzed for their in vitro binding properties, both constructs showed that only monomeric VP40 efficiently associated with membranes containing negatively charged lipids. Membrane association of truncated, hexameric VP40 was inefficient, indicating a membrane-recognition role for the C-terminal part. Based on these observations we propose that assembly of Ebola virus involves the formation of VP40 hexamers that is mediated by the N-terminal part of the polypeptide.

Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein.

Fur has been purified and reconstituted with Co2+ and Mn2+. The ESI-MS spectra of the apoprotein as well as Mn-Fur and Co-Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16,660 +/- 3 and 16,792 +/- 3 Da, which correspond, respectively, to the N-terminal methionine 'excised' or 'non-excised' forms of the monomer. This result proves the absence of any other post-translational modification or modification due to metal incorporation. On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal-containing forms in solution.

Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry.

To estimate the stability of Rhodobacter capsulatus ferrocytochrome c2 wild-type and site-directed mutants, charge state distributions and hydrogen/deuterium exchange rates were monitored by electrospray ionization mass spectrometry. The relative stability of the mutants was observed with the order: V11 insert > Y75F > wild-type = K32E > K12D = K14E > or = K52E > K14E/K32E > W67Y > P35A > I57N > G34S. (A preliminary account has been presented for mutants G34S and P35A [Jaquinod et al. (1995) Rapid Commun. Mass Spectrom. 9, 1135-1140].) This approach is shown to be a useful tool for rapid characterization of mutational effects on protein conformation.

Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning.

A new basic protein has been isolated from wheat endosperm by Triton X-114 phase partitioning. It contains five disulfide bridges and is composed of equal amounts of a polypeptide chain of 115 amino acid residues and of the same chain with a C-terminus dipeptide extension. The most striking sequence feature is the presence of a unique tryptophan-rich domain so that this protein isolated from wheat seeds has been named puroindoline. The similar phase partitioning behavior in Triton X-114 of this basic cystine-rich protein and of purothionins suggests that puroindoline may also be a membranotoxin that might play a role in the defense mechanism of plants against microbial pathogens.

New conformational properties induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough ferricytochrome c553 using isotopic exchanges monitored by mass spectrometry.

In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

Solution structure of glyceraldehyde-3-phosphate dehydrogenase from Haloarcula vallismortis.

The subunit molecular mass of glyceraldehyde-3-phosphate dehydrogenase from the extreme halophile Haloarcula vallismortis (hGAPDH) was determined by mass spectrometry to be 35990 +/- 80 daltons, similar to other GAPDHs. Complementary density, sedimentation and light scattering experiments showed the protein to be a tetramer that binds 0.18 +/- 0.10 gram of water and 0.07 +/- 0.02 gram of KCl per gram of protein, in multimolar KCl solutions. At low salt (below 1 M), the tetramer dissociated into unfolded monomers. This is the third halophilic protein for which solvent interactions were measured. The extent of these interactions depends on the protein, but all form an invariant particle, in multimolar NaCl or KCl solutions, that binds a high proportion of salt when compared to non-halophilic proteins.

Charnley total hip arthroplasty in patients less than fifty years old. A twenty to twenty-five-year follow-up note.

We evaluated the results twenty to twenty-five years after ninety-three consecutive, nonselected Charnley total hip arthroplasties performed with cement by the senior one of us in sixty-nine patients who were less than fifty years old at the time of the procedure. Seventy of the seventy-two hips in the living patients were followed radiographically for at least twenty years. Twenty-seven hips (29 per cent) had a revision or a resection of the prosthesis during the follow-up period. The revision or the resection was performed because of aseptic loosening in twenty-one hips (23 per cent), infection in four (4 per cent), dislocation in one (1 per cent), and fracture of the femur in one. Eighteen acetabular components (19 per cent) and five femoral components (5 per cent) were revised because of aseptic loosening, and an additional fourteen acetabular components (15 per cent) and seven femoral components (8 per cent) demonstrated definite or probable radiographic loosening. The present study demonstrates the long-term durability of total hip arthroplasty performed with cement in an active population of patients. The fixation of the femoral component was found to perform better than that of the acetabular component at twenty to twenty-five years after the procedure.

Explicit symplectic integrator for s-dependent static magnetic field.

This paper reports our recent work on explicit symplectic integration techniques for the charged particle motion in an s-dependent static magnetic field. Using the extended phase space, symplectic integrators can be developed for Hamiltonians with or without the paraxial approximation using either the space or time as an independent variable. This work extends the successful element-by-element tracking method for studying single-particle nonlinear dynamics to a set of s-dependent magnetic elements. Important applications of this work include the studies of the charged particle dynamics in a storage ring with various insertion devices, superconducting magnets, large aperture magnets with significant fringe fields, and solenoid magnets in the interaction region. Consequently, this work is expected to make an impact on design and optimal operation of existing and future light source rings and high energy physics accelerators.

[Value of extemporaneous pathology sections in cone biopsies]. / Intérêt de l'examen anatomopathologique extemporané dans les conisations.

OBJECTIVE: To assess the usefulness of frozen sections in cone biopsies. METHODS: Retrospective study of 60 cases: 26 without frozen section, from January 1985 to December 1988 (group 1); 34 with frozen section of the endocervical upper part of the cone, from January 1989 to December 1992 (group 2). RESULTS: The average height of the cone was 1.48 cm in group 2, and 2.23 cm in group 1. The cone margins were free of disease in 100% of cases in groupe 2, and in 88.5% in group 1. The rate of secondary hemorrage was 2.9%, compared with of 7.6% in group 1. CONCLUSION: Frozen section of endocervical upper part of the cone allows more economic excisions of healthy tissues as well as suppression of cases with involved cone margins. With technical improvement of electro-surgical units, we changed since 1992 from cold knife conization to large loop excision of the transformation zone, in association with frozen sections.

Maps for distributions and their time evolution.

Many dynamical stochastic processes occur "on top" of a deterministic process. We present a method which uses the trajectory of the deterministic process as basis functions for quasiarbitrary distributions. A map for the stochastic process can then be computed. This may have applications in electron storage rings or other devices perturbed by a small stochasticity. In this paper we will look only at the most elementary applications of the method.

Characterization of elemental sulfur in isolated intact spinach chloroplasts.

Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of (35)SO(4) (2-) resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S(8)) of elemental sulfur (S(0), valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S(0)) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S(8) under the experimental conditions used for analysis.

Peptide mapping and disulfide bond analysis of the cytoplasmic region of an intrinsic membrane protein by mass spectrometry.

Intrinsic membrane proteins pose substantial obstacles to analysis by common analytical techniques due to their hydrophobic nature and solubilization requirements. This is the case for studies involving HPLC coupled to mass spectrometry. We have developed an HPLC/mass spectrometry approach to explore and map the peptide sequence of the SERCA1a Ca(2+)-ATPase from the sarcoplasmic reticulum an integral membrane protein of 110 kDa. After extensive proteolysis of the protein, the mass of the proteolytic fragments was analyzed by HPLC/mass spectrometry. Only part of the cytoplasmic fragments was recovered under nondenaturing conditions. On the other hand, peptide fragments obtained under denaturing conditions were found to cover nearly all the cytoplasmic region. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase contains 24 cysteine residues, 18 of which are in the cytosolic or lumenal region of the protein. Peptides containing free cysteines were identified by a mass increase resulting from carboxyamidomethylation of the cysteines with iodoacetamide. Alkylation reactions were executed either before or after reduction of the peptide fragments by dithiothreitol. Analysis of the mass of the fragments indicates that no disulfide bonds exist in the cytoplasmic portion of SR Ca(2+)-ATPase.

Observation of holoprotein molecular ions of several ferredoxins by electrospray-ionization-mass spectrometry.

Several ferredoxins containing [4Fe-4S] or [2Fe-2S] active sites have been analyzed by electrospray-ionization-mass spectrometry. For these acidic proteins, low pH conditions must be implemented in order to ensure strong signals in positive-ionization mode. Under such conditions the iron-sulfur active sites were lost in most cases. In contrast, the holoproteins were preserved under negative-ionization mode conditions: they were weakly but sufficiently ionized and information about their cofactor content could be obtained. The experimental conditions set up here should provide a useful basis for the detailed characterization of more complex iron-sulfur proteins.

Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus.

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.

Electrospray-ionization mass spectrometry of molecular variants of a [2Fe-2S] ferredoxin.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum is a homodimeric protein of which each subunit contains one [2Fe-2S] cluster. In previous investigations, the five cysteine residues in positions 11, 14, 24, 56 and 60 had been mutated into serine or alanine. The wild type ferredoxin and several of its molecular variants have now been analyzed by electrospray-ionization mass spectrometry. In the negative-ion detection mode, depending on the infusion solvent used, molecular peaks attributable to the apoprotein, to the monomeric holoprotein, and to the dimeric holoprotein were detected in all cases. The data confirmed the presence of the expected mutations, showed that all of these proteins contain one [2Fe-2S] cluster per subunit, and indicated that the dimeric structure of these ferredoxins could be retained in the conditions of the electrospray ionization. This investigation establishes the power of electrospray-ionization mass spectrometry for the analysis of oligomeric proteins containing labile metal clusters.