Crystallization and preliminary x-ray diffraction study of the flavoprotein NADH peroxidase from Streptococcus faecalis 10C1

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.     

One thousand heart attacks in Grampian: the place of cardiopulmonary resuscitation in general practice.

The outcome of 1011 heart attacks in patients under the care of general practitioners who practised cardiopulmonary resuscitation and were equipped with defibrillators is reported. The 28 day mortality was 36% (367 patients), and 59% of deaths occurred outside hospital. The general practitioner was the first medical contact in 92% of heart attacks and was equipped with a defibrillator in 80% of such calls. Fifty six patients had a cardiac arrest in the presence of a general practitioner, and resuscitation was attempted in 47 cases, representing 5% of all calls for heart attacks. Twenty one (45%) resuscitated patients reached hospital alive, and 13 (28%) survived to leave hospital. The opportunities for cardiopulmonary resuscitation in general practice occur sufficiently often to warrant training and equipping general practitioners for advanced life support. The results of resuscitation by general practitioners working alone compare favourably with those of mobile coronary care units based in hospitals.     

Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.     

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.     

Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAF-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc2Ga14) or blood group A (Fuc2(Ga1NAc3) (Ga14) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the 3-linked G1cNAc at branch points, whereas the 6-linked GlcNAc residue ultimately forms short side chains with a Fuc2 (Ga1NAc3) Gal4 G1cNAc6 structure in individuals with A blood group determinant.The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. {ie75-1} This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of a2-linked fucose to the 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N , N -dimethyl- p -phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf ( p -dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes , and Corynebacterium . Of the various isolates, Alcaligenes DM₄ showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM₄ grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM₄ has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM₄. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.     

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.