Purification and characterization of S-formylglutathione hydrolase from human, rat and fish tissues.
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Animals↗
Biomedical subjects
R Lapatto
Publications and source records attributed to R Lapatto.
Nerve growth factor: structure/function relationships.
Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted beta-hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell-surface receptor types expressed in a variety of neuronal and non-neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site-directed mutagenesis, and this information can be related to their location in the 3-dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both promoters in the dimer.
Amino Acid Sequence↗
Nerve growth factor revisited.
Recent studies on nerve growth factor have revealed important new insights into the structure, function and evolution of this prototypical neurotrophic factor. Some of its features are (1) it has a unique three-dimensional fold that has since been found in two other growth factors, (2) it uses the trk proto-oncogene product, which has a tyrosine kinase, as a receptor and (3) it shares homology with at least three other factors, now collectively called neurotrophins, which have a spectrum of target cells.
Amino Acid Sequence↗
High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.
beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.
Amino Acid Sequence↗
New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor.
Nerve growth factor (NGF) is a member of an expanding family of neurotrophic factors (including brain-derived neurotrophic factor and the neurotrophins) that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous systems. Its biological effects are mediated by a high-affinity ligand-receptor interaction and a tyrosine kinase signalling pathway. A potential use for NGF and its relatives in the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease requires an understanding of the structure-function relationships of NGF. NGF is a dimeric molecule, with 118 amino acids per protomer. We report the crystal structure of the murine NGF dimer at 2.3-A resolution, which reveals a novel protomer structure consisting of three antiparallel pairs of beta strands, together forming a flat surface. Two subunits associate through this surface, thus burying a total of 2,332 A. Four loop regions, which contain many of the variable residues observed between different NGF-related molecules, may determine the different receptor specificities. A clustering of positively charged side chains may provide a complementary interaction with the acidic low-affinity NGF receptor. The structure provides a model for rational design of analogues of NGF and its relatives and for testing the NGF-receptor recognition determinants critical for signal transduction.
Amino Acid Sequence↗
Model for the structure of formaldehyde dehydrogenase based on alcohol dehydrogenase.
The three-dimensional structure of rat liver formaldehyde dehydrogenase (FALDH), previously known as class III alcohol dehydrogenase, was constructed using computer graphics and computer programs developed for model building. The construction is based on horse liver alcohol dehydrogenase (EE-ADH), whose structure has been elucidated by X-ray crystallography. The high sequence homology between the two enzymes makes knowledge-based modelling feasible in this case. The model shows a remarkable similarity to horse liver alcohol dehydrogenase especially in the NAD-binding domain. Certain mutations, and the one insertion in FALDH compared to EE-ADH in particular, have cause important changes in the substrate binding site, and thus aliphatic alcohols have been replaced by hemi-thioacetals as favourable substrates.
Alcohol Dehydrogenase↗
X-ray analysis of beta B2-crystallin and evolution of oligomeric lens proteins.
The beta, gamma-crystallins form a class of homologous proteins in the eye lens. Each gamma-crystallin comprises four topologically equivalent, Greek key motifs; pairs of motifs are organized around a local dyad to give domains and two similar domains are in turn related by a further local dyad. Sequence comparisons and model building predicted that hetero-oligomeric beta-crystallins also had internally quadruplicated subunits, but with extensions at the N and C termini, indicating that beta, gamma-crystallins evolved in two duplication steps from an ancestral protein folded as a Greek key. We report here the X-ray analysis at 2.1 A resolution of beta B2-crystallin homodimer which shows that the connecting peptide is extended and the two domains separated in a way quite unlike gamma-crystallin. Domain interactions analogous to those within monomeric gamma-crystallin are intermolecular and related by a crystallographic dyad in the beta B2-crystallin dimer. This shows how oligomers can evolve by conserving an interface rather than connectivity. A further interaction between dimers suggests a model for more complex aggregates of beta-crystallin in the lens.
Amino Acid Sequence↗
The 3-D structure of HIV-1 proteinase and the design of antiviral agents for the treatment of AIDS.
A proteinase is essential for replication of HIV. Cloning and chemical synthesis have provided a sufficient supply of HIV-1 proteinase for the determination of its three-dimensional structure. Analogies between the structures of HIV-1 proteinase and the mammalian enzyme renin, which is involved in the control of blood pressure, have given important clues concerning the design of specific inhibitors that have antiviral activity.
Acquired Immunodeficiency Syndrome↗
X-ray crystallographic studies on murine nerve growth factor.
The largest and best characterised family of neurotrophic growth factors is that of nerve growth factor (NGF) and its relatives. In order to understand the relation of structure and function, we have undertaken X-ray analyses of murine NGF. The active component beta-NGF crystallises as hexagonal bipyramids that give good X-ray diffraction data using a synchrotron to 2.3A resolution. We have prepared several heavy atom derivatives that are being used in the method of multiple isomorphous replacement to solve the phase problem and determine the three-dimensional structure. We have also prepared crystals of the precursor, 7S NGF, which is a complex of three different subunits of composition alpha 2 beta 2 gamma 2. We have collected X-ray data to 3A resolution on two crystal forms with related cell dimensions and orthorhombic spacegroups. Detailed analyses of the structures of NGF in these crystal forms, taken together with data on sequence and biological activity, should give clues concerning the role of the precursor complex in storage and assist the identification of the surface region involved in receptor binding.
Amino Acid Sequence↗
X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes.
Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.
Crystallography↗
Quantitative trace element analysis of human nails with external beam PIXE.
External beam PIXE (Particle Induced X-ray Emission) analysis with a proton beam of 2.4 MeV was used to study trace element concentrations in human nails. The suitability of PIXE analysis regarding nail samples without any pretreatment besides washing was investigated. The main emphasis has been on the ability to obtain absolute concentration values and a new accurate method for nail sample standardization has been developed. Concentration values for the elements Ca, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, and Pb were determined from human nail samples. A comparison was made with nail samples taken from different fingers and toes to monitor intraindividual variation, and nails of different healthy individuals to get a view of the interindividual differences. The concentrations were also measured in relation to time in order to observe any possible short-term changes. The results are compared with the previous studies reported in the literature. The nail analysis is also compared to hair analysis in terms of detection limits, number of elements determinable, and standardization of the results.
Humans↗
Placental alcohol metabolism in chronic alcohol abuse.
The activities of aldehyde dehydrogenase (ALDH; EC 1.2.1.3) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were measured in term placentas of 13 alcoholic women and 16 matched controls. With acetaldehyde 8 mmol/l as substrate, the ALDH activity was 29.1 +/- 12.2 and 34.4 +/- 15.3 mU/g of wet weight (mean +/- SD; p greater than 0.4) for alcoholics and controls, respectively. With 50 mumol of acetaldehyde, ALDH activity was undetectable in both groups. No ADH activity could be detected in the placentas. The weights of placentas and newborns were significantly lower in the alcoholic group (placentas: 526 +/- 116 vs. 653 +/- 77 g, p less than 0.005; newborns 2,878 +/- 417 vs. 3,595 +/- 346 g, p less than 0.001). The results suggest that in chronic alcohol abuse, the placenta plays a negligible role in the metabolism of ethanol and acetaldehyde.
Acetaldehyde↗
A rapid method for the quantification of C, N and O in biomedical samples by proton-induced gamma-ray emission analysis applied to human placental samples.
A new fast, accurate and simple quantification method for C, N and O in biomedical samples by external-beam proton-induced gamma-ray emission analysis is presented. Concentration values for C, N and O in commercial standards of NBS bovine liver 1577a, IAEA animal muscle H-4 and IAEA horse kidney H-8 were determined using the new method. The concentrations of C, N, O, Na, Mg and P were determined from placental samples of 13 alcoholic mothers and from 16 abstinent controls. The values obtained for C, N, O, Mg and P were the first values ever obtained for human placentas.
Alcoholism↗
Trace element studies on whole human cerebrospinal fluid with external beam PIXE.
External beam PIXE analysis with a 2.4 MeV proton beam was used to determine the concentrations of K, Ca, Fe, Cu, Zn and Br in cerebrospinal fluid from patients having various disorders. The obtained total concentration ranges K 34,000-1,079,000, Ca 5300-81,300, Fe 40-1030, Cu 20-1650, Zn 15-1250 and Br 400-43,000 micrograms/kg are compared with the values given in the literature. In certain patients there were very high CSF bromine levels, but this was shown to be the result of taking medications presented as bromide salts. The possibility of using the method in clinical practice for CSF analysis is considered. The new method of preparing self-supporting films of the samples was used. This method was further optimized by investigating in detail the use of EDTA as a homogenizer.
Adolescent↗
Preparation of thin self-supporting samples of liquids for external beam PIXE analyses: application to human cerebrospinal fluid.
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Bromides↗