Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction, Overexpression, and Purification of Arthrobacter globiformis Amine Oxidase–Strep-Tag II Fusion Protein

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.     

The microbiology of cut-away peat

Summary Fungi occurring commonly in cut-away peat were selected for autecological study, being chosen to represent both cosmopolitan and indigenous elements of the peat flora. A fungus alien to peat,Fusarium culmorum, was also included in the study. The germination in peat of the spores of these species and their ability to grow and colonise organic material added to peat were examined. It was concluded that the cosmopolitan element was composed of efficient saprophytes but that the ability of their spores to germinate in unamended peat was limited. Although Fusarium spores germinated, the germ tubes subsequently formed chlamydospores. Only spores of the indigenous peat fungi germinated well in peat but their growth habit was not suited to the colonisation and utilisation of organic matter, added to peat, in competition with the cosmopolitan flora. The concept of ‘source potential’ is introduced to explain the variance in fungal growth from different substrate bases.     

Differential decrease of the central beta-adrenergic receptor in the rat after subchronic infusion of desipramine and clenbuterol

The effect of subchronic infusion of desipramine, a norepinephrine uptake inhibitor, and clenbuterol, a beta-adrenergic agonist, on the central beta receptor of the rat was determined using in vitro [³H]dihydroalprenolol binding. Desipramine produced significant decreases of the receptor in neocortex and hippocampal formation, and clenbuterol effected such decreases in corpus striatum and cerebellum. Both drugs caused a marked decrease in the activity of isoproterenol-sensitive adenylate cyclase in neocortex. The alpha₂ receptor of neocortex and cerebellum was unchanged by either drug as assessed by in vitro$\text{[}{}^3\text{H}\text{]p-}\text{aminoclonidine}$ binding. The results are discussed in terms of the different mechanisms of action of desipramine and clenbuterol, and the efficacy of these two drugs in the treatment of depression.     

Development of rabbit embryos during a 96-h period of in vitro culture after superovulatory treatment under conditions of elevated ambient temperature.

The effects of elevated ambient temperature on the response to exogenous gonadotropins were evaluated in female New Zealand White rabbits exposed to 33+/-1 degrees C (mean +/- SE) and 10-30% relative humidity (8 h/day) during a 5-day period. Does were treated with pFSH (0.3 mg/0.3 ml Standard Armour) twice daily during three consecutive days with a minimum interval of 8 h between injections. Six hours after the last FSH injection all does were removed from the experimental chamber, given hCG (25 IU/kg) and paired overnight. Nineteen hours after pairing, embryos were flushed from the reproductive tracts, evaluated, and subjected to in vitro culture during a 96-h period. The ovulatory responses to exogenous gonadotropins and fertilization rates did not differ significantly under conditions of elevated ambient temperature, whereas fewer blastocysts and increased number of degenerate embryos were observed after culture. We conclude that although hyperthermia was induced during exposure to elevated ambient temperature, it did not alter the ovulatory responses to gonadotropin treatment and plasma concentrations of FSH and LH compared with does in a thermoneutral environment. Exposure of donor rabbits to elevated ambient temperature before mating, however, increased embryonic degeneration.