A reassessment of the MAdCAM-1 structure and its role in integrin recognition.

Mucosal addressin cell-adhesion molecule (MAdCAM-1) is a membrane-bound leukocyte receptor regulating both the passage and retention of leukocytes in mucosal tissues. A crystal structure for the two extracellular amino-terminal domains of human MAdCAM-1 has previously been reported, confirming their expected immunoglobulin superfamily topology. In this study, a second crystal structure of this fragment is described. Although the overall structure is similar to that previously reported, one edge strand in the amino-terminal domain is instead located on the opposite sheet. This alters the arrangement and conformation of amino acids in this region that have previously been shown to be crucial for ligand binding. MAdCAM-1 is also seen to form dimers within the crystal lattice, raising the possibility that oligomerization may influence the biological role of this adhesion molecule.

Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase.

Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.

Crystallization of cytochrome b562 from Erwinia chrysanthemi.

Cytochrome b(562) from Erwinia chrysanthemi has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. X-ray precession photographs show that the crystals formed belong to either of the enantiomorphic space groups P4(1)2(1)2 or P4(3)2(1)2 with the cell parameters a = b = 98.6 and c = 62.7 A. Estimation of the crystal density and consideration of the possible values for V(m) indicate that there is either a dimer or trimer in the asymmetric unit. Experiments using the synchrotron radiation source at the CCLRC Daresbury Laboratory have shown that the crystals diffract to at least 2.7 A resolution. An analysis of the N-terminal sequence indicates that this cytochrome shows limited homology to the cytochrome b(562) from E. coli. Determination of the structure will therefore allow analysis of the relationship between these two proteins.

Crystallization of glycerol dehydrogenase from Bacillus stearothermophilus.

The NAD(+)-linked glycerol dehydrogenase from Bacillus stearothermophilus is a member of the so-called 'iron- containing' class of polyol dehydrogenases. This enzyme has been crystallized in three different forms in the presence of a range of divalent cations and glycerol or NAD+ using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. X-ray photographs have established that the crystals grown in the presence of zinc and glycerol (form A) most likely belong to space group I4(1)22 with cell parameters a = b = 102 and c = 728 A. The crystals grown with zlnc and NAD(+) (form B) belong to the tetragonal system and probably belong to the space group P42(1)2 with cell parameters a = b = 150 and c = 220 A. The crystals grown with lead and glycerol (form C) belong to a primitive orthorhombic system with cell parameters a = 127, b = 178 and c = 173 A. Experiments using the synchrotron radiation source at the DRAL Daresbury laboratory have shown all three crystal types diffract to at least 3 A resolution. Elucidation of the three-dimensional structure of this enzyme will provide a structural framework for this class of polyol dehydrogenases, which are not represented in the database at present, and enable comparisons to be made with enzymes belonging to the other classes.

Crystallization and analysis of the subunit assembly and quaternary structure of imidazoleglycerol phosphate dehydratase from Saccharomyces cerevisiae.

Imidazoleglycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae has been crystallized in the presence of a range of divalent cations using the hanging-drop method of vapour diffusion with ammonium sulfate or polyethylene glycol (PEG) 4000 as the precipitants. X-ray precession photographs have established that the crystals formed with ammonium sulfate (form A) belong to the space group F432, with cell parameter a = 177.5 A and a single subunit in the asymmetric unit. A preliminary data set collected to 6 A resolution on a two-detector San Diego Multiwire area detector has established that the crystals formed with PEG 4000 (form B) belong to either of the special pair of space groups I23 or I2(1)3, with cell parameter a = 131.0 A. A self-rotation function has been calculated using these data and indicates that the cell axes show pseudo fourfold symmetry consistent with a dimer in the asymmetric unit in this crystal form. Light-scattering studies indicate that in the presence of Mn(2+) and a number of other divalent cations IGPD undergoes assembly to a particle of molecular weight approximately 500 kDa. Given the subunit molecular weight of 23 kDa together with the symmetry of the crystals it would indicate that the most likely quaternary structure for this enzyme is based on a 24-mer in 432 symmetry.

Purification and characterization of the imidazoleglycerol-phosphate dehydratase of Saccharomyces cerevisiae from recombinant Escherichia coli.

The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

The use of transabdominal ultrasound in the detection of early bladder tumours.

We have studied the accuracy of transabdominal ultrasound (TAUS) in the diagnosis of early superficial bladder carcinoma. One hundred and twenty-six patients returning for check cystoscopy were scanned pre-operatively with the Technicare Autosector Scanner and the results compared with the endoscopic findings. Forty-four patients (37%) had a tumour recurrence. TAUS demonstrated a lesion in 50% of these with a false positive rate of 11%. The diagnostic accuracy was proportional to the tumour size (82% of patients with tumours above 5 mm were detected compared with 38% below 5 mm), but was not affected by grade, stage or position in the bladder. TAUS may prove a useful adjunct to cytology as a screening test for bladder cancer.