Enhanced detection of glycoproteins in polyacrylamide gels

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.     

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.     

5'-Methylbenzimidazolyl-cobamides are the corrinoids from some sulfate-reducing and sulfur-metabolizing bacteria

The sulfate-reducing bacteria Desulfobacterium autotrophicum, Desulfobulbus propionicus and Archaeoglobus fulgidus (VC-16) and the sulfur-metabolizing archaebacteria Desulfurolobus ambivalens and Thermoplasma acidophilum were found to contain considerable amounts of corrinoids, that were isolated and crystallized in their Co beta-cyano form. In three other sulfur-metabolizing archaebacteria, Thermoproteus neutrophilus, Pyrodictium occultum and Staphylothermus marinus significant amounts of corrinoids were not detected under the isolation methods used. The samples from the three sulfate-reducers were identified with Co alpha-[alpha-(5'-methylbenzimidazolyl)]-Co beta-cyanocobamide. This corrinoid was also obtained from a 5-methylbenzimidazole-supplemented Propionibacterium fermentation and was structurally characterized by ultraviolet/visible, CD, fast-atom-bombardment MS, 1H-and 13C-NMR spectroscopy. Also the major corrinoid from T. acidophilum was (tentatively) analyzed as a 5'-methylbenzimidazolyl-cobamide, whereas the main corrinoid from D. ambivalens was indicated to be vitamin B12 (a 5',6'-dimethylbenzimidazolyl-cobamide). The 5'-methylbenzimidazolylcobamides are found here as the common corrins of some sulfate-reducing and sulfur-metabolizing bacteria. The structural diversity due to the differing nucleotide bases of the corrins examined here and in methanogenic and acetogenic bacteria appears not to correlate to the biological function(s) of the corrins, but rather to be determined by biosynthetic properties of these organisms under natural growth conditions.     

Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion-exchange chromatography on DEAE-cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high-molecular-mass (P-HS 1) and a low-molecular-mass (P-HS 2) fraction by gel filtration on Sephacryl S-400. P-HS 1 has a Mr of 175,000 and possesses four heparan sulfate side-chains (Mr 32,000) covalently bound to the protein core via a galactose- and xylose-containing polysaccharide-protein binding region. The protein core (Mr 38,000), which was obtained after deglycosylation of PG-HS 1 with trifluormethane sulfonic acid, contained in addition a few N-glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P-HS 2 is a single-chain peptidoheparan sulfate of Mr of 38,000 containing one heparan sulfate chain (Mr 32,000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P-HS 1 and P-HS 2 was 1:0.66.     

Rapid-scan stopped-flow studies of the flavoenzyme mercuric reductase during catalytic turnover

Time-resolved absorption spectra of the FAD-containing enzyme mercuric reductase were recorded during the catalytic reaction at 25 degrees C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k = 43 s-1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two-electron-reduced enzyme, EH2-NADPH. This spectrum persisted during the quasisteady-state phase of the reaction suggesting that EH2-NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2-NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2-NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two-electron-reduced enzyme, EH2-NADP+. These results suggest that EH2-HADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3-40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, less than 1.5 ng/ml (range less than 0.9-3); liver cirrhosis less than 1.5 ng/ml (range less than 0.9-38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54-477); ileal resection 397 ng/ml (range 128-750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.     

Interaction of site specific hirudin variants with alpha-thrombin

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher Ki value for their complexes with thrombin. The observed effects are mainly due to increased koff rate constants.     

Trypanosoma cruzi: origin of metacyclic trypomastigotes in the urine of the vector Triatoma infestans

Population density and percentage of the different stages of an established infection of Trypanosoma cruzi were determined for two parts of the excretory system and for the rectum of fifth instars of Triatoma infestans unfed and 4 hr after feeding. These data were also evaluated for feces and urine of the fed bugs. In the first unfed group only small populations of the flagellate occurred in the Malpighian tubules and ampullae and not in all bugs. The three rectal populations (rectal lumen and anterior and posterior rectal wall) consisted of approximately equal numbers. About 10% were spheromastigotes and about 10% were stages intermediate to epimastigotes. Significantly fewer epimastigotes and more trypomastigotes were present on the rectal wall than in the lumen. Two intermediate forms leading to the trypomastigote stage occurred in similar numbers. In nearly all bugs the initial excretion (feces) contained the highest number of flagellates as compared to the following drops of urine. More flagellates were excreted through the urine than were contained in the excretory system of unfed bugs. The population in the feces reflected the percentage of forms present in the rectal lumen of unfed bugs, but in the urine the percentage of trypomastigotes increased up to 100%. Four hours after blood uptake, dissection of bugs still showed parasites in the Malpighian tubules and ampullae; the total number of parasites in the rectum was reduced by more than 50%. This reduction was more pronounced in the rectal lumen and on the posterior rectal wall. In stained smears from all three rectal populations there were rarely spheromastigotes but high percentages of epimastigotes. The intermediate stages leading to trypomastigotes mainly originated from short epimastigotes. Comparison of the T. cruzi populations before and after feeding demonstrates that the trypomastigotes in the urine should originate from the rectal wall, especially from the posterior part.     

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.