The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FBPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Reaction of oxygen with cytochrome c oxidase from Paracoccus denitrificans

The reaction of reduced cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans (American Type Culture Collection 13543) with dioxygen has been followed by laser flash photolysis of the CO derivative. In detergent-stabilized solutions the reaction showed at least two distinct kinetic components, the faster of which was oxygen concentration dependent and had a rate of approximately 60 X 10(6) M-1 s-1. The slower reaction was independent of oxygen concentration and had a rate of 9 X 10(2) s-1. These rates are about 1.5 times greater than comparable rates for ox heart oxidase reported by C. Greenwood and Q. H. Gibson (J. Biol. Chem. (1967) 242, 1782-1787). The kinetic components have markedly different optical spectra which agree precisely in form with those for ox heart enzyme (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787) but are shifted by 2 nm toward the red. In phospholipid vesicles, the spectral contribution of the faster component was augmented. The dissociation constant for CO at 20 degrees C is 1.6 microM, 6 times greater than for the ox heart enzyme. The bacterial enzyme binds one CO per 2 heme a. The enzyme has an absorption band at 830 nm in the oxidized form similar to that of the ox heart enzyme.     

Algal oxidation of aromatic hydrocarbons: formation of 1-naphthol from naphthalene by Agmenellum quadruplicatum, strain PR-6.

Hydroxyurea induces repair replication in human lymphoblastoid NC37 BaEV cells during incubation with liver microsomes and NADPH. Catalase reduces hydroxyurea-induced repair by more than 95%, suggesting that hydrogen peroxide derived from hydroxyurea in the presence of the metabolic activation system is involved in the DNA damage.     

A useful method for the analysis of growth studies

A group of parsimonious models for the analysis of animal growth curves is presented. The effects of correlations between the successive weights are reduced by analyzing the weight gained between the different weighing times. A model is developed for the resulting two-way table of group average gain curves (group by periods), using the two-way table modeling techniques proposed by Mandel. The coefficients in the fitted model are examined to determine what functions of the weight gain measurements will summarize the characteristics of the curves. In the second part of the analysis these statistics are computed for each animal and analyzed to determine the effects of the design variables on the growth of the animals. Experience has shown that in many instances total weight gain summarizes all the information in the curve. The conditions under which total gain provides an adequate summary are discussed. The model has been used in the analysis of rat, dog, hamster, guinea pig and swine growth curves. An example is included to illustrate the utility of the proposed approach.     

Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

The measurement of partial specific volumes and sedimentation coefficients by sucrose density centrifugation

The method of Martin and Ames (1961, J. Biol. Chem.236, 1372) gives good estimates of the s_20,w of proteins when the SW40 rotor is used with a sucrose gradient. Viscosities of sucrose in D₂O were measured, and the data were used in computer simulations to test alternate approaches to estimating $\text{v}$ and s_20,w values by comparisons with standards. The method of Meunier et al. (1972, FEBS Lett.24, 63) for $\text{v}$ was shown to be optimal. For s_20,w estimations, substantial errors were found with the methods of Bon et al. (1973, Eur. J. Blochem.35, 372) and especially Meunier et al. When standards and unknowns have the same $\text{v}$, and the gradient is made up in water or dilute buffer, the simple ratio method of Martin and Ames gives most accurate results for s_20,w. For all other cases, an alternative procedure is described.