Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.     

Isolation and mapping of ribosomal RNA genes of Caulobacter crescentus

Ribosomal DNA fragments of 1.0, 3.4, 3.7 and 6.1 kb² produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RNA probe. These genomic sequences were further characterized by the isolation of 13 hybrid λ Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Cc1880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to ³²P-labeled rRNA. Thus, the two clones contain different DNA inserts which together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S-5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 × 10⁹ daltons. The relatively simple organization of rDNA sequences in the C. crescentus chromosome compared to Escherichia coli is discussed.     

Isolation and identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro metabolites of all-trans-retinoic acid in hamster trachea and liver.

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.     

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.     

Purification and characterization of a polyhook protein from Caulobacter crescentus.

A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.     

Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet stage.