The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Structure of the fluorescent nucleoside of yeast phenylalanine transfer ribonucleic acid

The structure of enzymatically isolated Y nucleoside of yeast phenylalanine tRNA was established by comparing its absorption, fluorescence, and mass spectra to that of the free base. The site of ribosylation was tentatively deduced by comparing the behavior under acid conditions of the natural nucleoside to that of synthetic Y nucleoside analogs. Our results indicate that the aglycone of the enzymatically isolated nucleoside has the same structure as the free base excised by acid treatment of phenylalanine tRNA, and that the ribose is probably attached to the N-3 position of the tricyclic nucleus.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.     

Inductive activity and enduring cellular constitution of a supernumerary apical ectodermal ridge grafted to the limb bud of the chick embryo.

Apical ectodermal ridges (AERs) isolated from 3- to 4-day chick and quail embryos were prepared by means of trypsinization and microdissection and then were grafted to the dorsal or ventral side of a host chick wing bud. They induced supernumerary limb outgrowths from the host bud showing, respectively, a bidorsal or biventral organization, as determined by the patterns of feather germs. The grafted ridge cells persisted, as revealed by histological sections of supernumerary chick limb parts growing under the influence of quail AERs, whose cells are readily distinguished after application of the Feulgen reagent.These results show that the AER induces limb outgrowth regardless of whether it is associated with dorsal or ventral limb ectoderm and that its continued existence is not dependent on contributions of ectodermal cells from the opposed ectodermal faces of the limb bud. The AER is pictured as maintaining the subjacent mesoderm in a condition of developmental plasticity without specifying its differentiation with respect to the proximodistal axis. It remains uncertain whether the positional values of cells that develop under the influence of the AER arise within these cells themselves or appear in response to influences from proximal sources.     

Relationship between abundance, growth, egg size and fecundity in a landlocked population of longfin smelt, Spirinchus thaleichthys

Sixteen year-classes of longfin smelt, Spirinchus thaleichthys , show a consistent pattern of alternating year-class strength between even (strong) and odd (weak) years. A recent (1989) odd year-class grew as well as, if not better than, the late 1960s odd year-classes. However, fish in the even year-classes (1988 and 1990) were significantly smaller in size then the 1966 and 1968 cohorts. Associated with this reduction in growth of the even year-class fish is a decline in fecundity. Fecundity of the odd year-classes of smelt was also smaller than in the early 1960s. Mean egg diameter and gonadosomatic index were not statistically different between 1988 and 1989 year-classes but the relative fecundity was higher for the 1988 year-class than for 1989, suggesting a higher reproductive investment in the even year-class. Reduction in growth and associated reproductive traits is considered to result from a reduction in the abundance of mysids, a preferred prey species.     

Interaction of cobalt(II) bovine carbonic anhydrase with pyridine-2,6-dicarboxylate ion and other chelating ligands

The removal of cobalt from cobalt(II) bovine carbonic anhydrase by pyridine-2-carboxylate, pyridine-2,6-dicarboxylate and 5-methyl-1,10-phenanthroline occurs via formation of an intermediate. This is presumed to be a ternary adduct of cobalt(II) enzyme with the ligand. In this, metal-protein bonds are loosened, probably via distortion of the normal geometry, resulting in accelerated breakdown of the adduct to apoprotein, compared with the behavior of the cobalt(II) enzyme alone. With 2-carboxy-1,10-phenanthroline, removal of metal is very rapid but no adduct is observed. Values of stability constants of the adducts and rate constants for their decomposition to apoprotein and their formation from apoprotein and cobalt(II) complex were measured at pH 5.5 and 25°C. Formation and dissociation rate constants for the adduct of cobalt carbonic anhydrase with pyridine-2,6-dicarboxylate could be measured from pH 5 to 7 and 10° to 25°C by stopped flow. Values of thermodynamic parameters for the various reactions agreed well with those estimated from the kinetic data.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

Implementing Extended Producer Responsibility Legislation

The goal of this article is to contribute to the understanding of how the multiple, and sometimes conflicting, stakeholder perspectives and prevailing conditions (economic, geographic, etc.) in the implementation locality shape extended producer responsibility (EPR) “on the ground.” We provide an in‐depth examination of the implementation dimension of EPR in a specific case study by examining concrete activities at the operational front of the collection and recycling system, and probing the varying stakeholder preferences that have driven a specific system to its status quo. To this end, we conduct a detailed case study of the Washington State EPR implementation for electronic waste. We provide an overview of various stakeholder perspectives and their implications for the attainment of EPR policy objectives in practice. These findings shed light on the intrinsic complexity of EPR implementation. We conclude with recommendations on how to achieve effective and efficient EPR implementation, including improving design incentives, incorporating reuse and refurbishing, expanding product scope, managing downstream material flows, and promoting operational efficiency via fair cost allocation design.     

Late Ordovician brachiopods from eastern North Greenland: equatorial offshore migration of the Red River fauna

Abstract: Late Ordovician rhynchonelliformean brachiopods, typical of the North American Red River fauna, are found sporadically in the Børglum River Formation of the Centrum Sø area, Kronprins Christian Land, eastern North Greenland. The geographical distribution of this characteristic brachiopod fauna is thus extended to the easternmost extremity of the Laurentian craton. The assemblage compares specifically with the Hiscobeccus brachiopod fauna, based on key taxa such as notably Hiscobeccus gigas (Wang, 1949), and indicates a late Katian age for this part of the succession. For the first time, this typically inland, shallow‐water fauna is found associated with genera like Bimuria, suggesting a transitional marginal facies with outer shelf benthos. The current study describes a Hiscobeccus fauna that lived at the seaward edge of its preferred habitat. Furthermore, an unpublished Hiscobeccus fauna, from the Børglum River Formation of Peary Land, central North Greenland, as well as several occurrences from the Kap Jackson and Cape Calhoun formations in various parts of Washington Land, western North Greenland, are described here. These show a distinct shift from older strata containing H. capax (Conrad, 1842) to younger strata exclusively yielding specimens of H. gigas. As H. gigas occurs in the upper part of the Cape Calhoun Formation in Washington Land, it indicates that the upper boundary of the Cape Calhoun Formation is considerably younger than previous estimates, reaching into the uppermost Katian (middle Cautleyan–Rawtheyan). The Cape Calhoun Formation correlates with the upper member of the Børglum River Formation and further demonstrates that the Hiscobeccus fauna was widespread in Laurentian marginal settings of North Greenland. Even though the Hiscobeccus fauna was pan‐continental during the late Katian (Richmondian), it possesses a strong provincial signal during the later Ordovician. The new occurrences indicate that this fauna extended to the north‐eastern margin of the Laurentian Craton. It lived in close association with cosmopolitan faunal elements that may have been the earliest sign of the succeeding invasion of migrants from Baltica that arrived later during the Hirnantian. The offshore migration of this atypical Hiscobeccus fauna likely demonstrates the path of warm‐water currents as the Centrum Sø locality was located at the equator during the Late Ordovician.     

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

In vivo, collagen I, the major structural protein in human body, is found assembled into fibrils. In the present work, we study a high concentrated collagen sample in its soluble, fibrillar, and denatured states using one and two dimensional {¹H}-¹³C solid-state NMR spectroscopy. We interpret ¹³C chemical shift variations in terms of dihedral angle conformation changes. Our data show that fibrillogenesis increases the side chain and backbone structural complexity. Nevertheless, only three to five rotameric equilibria are found for each amino acid residue, indicating a relatively low structural heterogeneity of collagen upon fibrillogenesis. Using side chain statistical data, we calculate equilibrium constants for a great number of amino acid residues. Moreover, based on a ¹³C quantitative spectrum, we estimate the percentage of residues implicated in each equilibrium. Our data indicate that fibril formation greatly affects hydroxyproline and proline prolyl pucker ring conformation. Finally, we discuss the implication of these structural data and propose a model in which the attractive force of fibrillogenesis comes from a structural reorganization of 10 to 15% of the amino acids. These results allow us to further understand the self-assembling process and fibrillar structure of collagen.