Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

HPLC shadowing: Artifacts in peptide characterization monitored by RIA

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with ¹²⁵I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.     

Thyroglobulin structure-function: the effect of iodination on the structure of human thyroglobulin

Thyroglobulin of very low iodine content has been prepared from a single non-toxic human goitre. The initial iodine content of the protein (0.038%) has been increased to levels of 0.16% and 0.85% by in vitro treatment with thyroid peroxidase and the resulting proteins studied with respect to their intrinsic fluorescence, circular dichroism spectra and binding of the hydrophobic probe 1,8-anilinonaphthalene sulfonic acid (ANS). While significant differences were observed between levels of iodination in both the ANS binding and intrinsic fluorescence of the thyroglobulin, no significant differences in the near and far UV circular dichroism spectra of the protein as a function of iodine content were observed. These data suggest that, the iodination of thyroglobulin effects specific areas of the protein without significant disruption of its overall secondary structure.     

Familial pericentric inversion of the X chromosome [inv(X)(p11q28)]

A familial pericentric inversion of the X chromosome [46,X,inv(X)(p11q28)] and [46,inv(X)(p11q28), Y] is reported. The carriers of the inv(X) presented no clinical symptoms. Either the inverted or the normal X chromosome may be late replicating.     

Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture

Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H₂/CO₂ or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an acetate oxidizer in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F₄₂₀ autofluorescence. The coculture completely degraded 40 mol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH₄. Studies with ¹⁴C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO₂ by the coculture and that CO₂ was concurrently reduced to CH₄. During growth, there was significant isotopic exchange between CO₂ and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO₂ and H₂ by one organism, while H₂ was subsequently used by a second organism to reduce CO₂ to CH₄. Since the H₂ partial pressure must be maintained below 10^-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936.     

Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli.

Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C). Additional initial-rate measurement were performed to assess specific points. Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves. Two models, both extensions of the concerted model given by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88--118] with four protomers, could be fitted to the data within the experimental error. Model discrimination in favour of one of these models was possible by proper experimental design. In the selected model one conformational state of the enzyme forms the active complex. The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations. In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site.     

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3''-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.