Calcium is required for the reduction of sulfite from hydrogen in a reconstituted electron transfer chain from the sulfate reducing bacterium, Desulfovibrio gigas

Calcium is found a strong stimulator of sulfite reduction from hydrogen. A coupling protein of molecular weight 65,000 can be isolated from Desulfovibrio gigas. It functions in a reconstituted electron transfer chain between hydrogenase and sulfite reductase. Its N-terminal sequence shows high homologies with calcium or magnesium binding sites from other calcium-binding proteins.     

APGW-amide as an inhibitory neurotransmitter of Achatina fulica Ferussac

APGWamide (L-Ala-L-Pro-Gly-L-Trp-NH2) was purified from the ganglia of an African giant snail (Achatina fulica Ferussac). This peptide inhibited (hyperpolarized) more than half of the Achatina neurone types tested. This produced an outward current with the membrane conductance increase of RAPN (right anterior pallial neurone) under voltage clamp. The ED50 of the peptide was 6.2 x 10(-6) M (95% confidence limit: 5.0-7.8 x 10(-6) M) and the Emax was 3.9 +/- 0.2 nA. The effects were due to a membrane permeability increase to K+. The peptide is proposed as an inhibitory neurotransmitter of the Achatina neurones.     

Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes.

The amino acid sequence of the copper-containing nitrite reductase (EC 1.7.99.3) from Achromobacter cycloclastes strain IAM 1013 has been determined by using peptides derived from digestion with Achromobacter protease I (Lys), Staphylococcus aureus V8 protease (Glu), cyanogen bromide, and BNPS-skatole in acetic acid. The subunit contains 340 amino acids. The identity of the first seven amino acids is tentative. The sequence has been instrumental in the X-ray structure determination of this molecule; in conjunction with the X-ray structure, ligands to a type I copper atom and a type II copper atom (one of each per subunit) have been identified. Comparison of the sequence to those of multi-copper oxidases such as ascorbate oxidase, laccase, and ceruloplasmin [Messerschmidt, A., & Huber, R. (1990) Eur. J. Biochem. 187, 341-352] reveals that each of two domains seen in the X-ray structure is similar to the oxidases and also to the small blue copper-containing proteins such as plastocyanin. The combination of sequence and structural similarity to ascorbate oxidase and sequence similarity to ceruloplasmin leads to a plausible model for the domain structure of ceruloplasmin.     

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of substrate and transition-state analogue complexes in crystals of phosphoglucomutase after removing the crystallization salt.

Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

Colorimetric determination of L-phenylacetyl carbinol produced by biotransformation of benzaldehyde and pyruvate using Saccharomyces cerevisiae

Summary The utility of a colorimetric method, based on the Voges-Proskauer reaction, for L-phenylacetyl carbinol (L-PAC) determination has been evaluated. The relative absorbances observed for equivalent concentrations of L-PAC and acetylbenzoyl at 580 nm were determined. L-PAC manifested a lower absorbance (60–68%) than the equivalent acetylbenzoyl concentration.