Circular dichroism of reduced and oxidized recombinant human epidermal growth factor.

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mM sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of alpha-helix, beta-sheet, and unordered structure. The addition of the alpha-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of alpha-helix, at the apparent expense of beta-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.     

Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function.

Plant alpha dioxygenases (PADOX) convert fatty acids to 2-hydroperoxy products that are important in plant signaling pathways. The PADOX amino-terminal domain is distinct from that in other myeloperoxidase-family hemoproteins, and the positional specificity and prosthetic group of PADOX distinguish them from the non-heme iron plant lipoxygenases. The constraints of the PADOX active site on potential substrates are poorly understood and only limited structure-function and mechanistic information is available for these enzymes. We developed several bacterial and insect cell systems for expression of recombinant Arabidopsis thaliana PADOX1 and evaluated the enzyme's substrate and inhibitor profiles and explored the functional role of the amino-terminal domain. Substrate specificity studies gave the following relative oxygenase activity values: linolenate, 1.00; linoleate, 0.95; oleate, 0.84; palmitoleate, 0.69; myristate, 0.23; palmitate, 0.17; and gamma-linolenate, 0.16. Methyl esters of myristate, linoleate and linolenate were not oxygenated. 3-Thiamyristate was the only oxygenase substrate that produced pronounced enzyme self-inactivation during catalysis. 3,4-Dehydromyristate inactivated the oxygenase without appreciable oxygen consumption. Several compounds inhibited oxygenase activity, including catechol (K(i) approximately 90 microM), divalent zinc ion (K(i) approximately 50 microM), N,N,N',N'-tetramethyl-p-phenylenediamine (K(i) approximately 20 microM) and cyanide ion (K(i) approximately 5 microM). Zinc ion did not change the K(m) values for linoleate or oxygen, or the K(i) value for cyanide, indicating that zinc acts at a distinct site from the other compounds. Gel-filtration chromatography revealed considerable variation in oligomeric state of recombinant PADOX1 produced in the various expression systems, but oligomeric state was not correlated with activity. Deletion of the first eight or fourteen PADOX1 residues in a NuSA-PADOX1 fusion protein led to 13 and 83% decreases in activity, respectively, indicating the N-terminal region is important for normal catalytic activity.     

Characteristic gamma-lactone odor production of the genus Pityrosporum.

Mass spectrometric-gas chromatographic analysis of culture headspaces revealed that members of the genous Pityrosporum produce volatile gamma-lactones during growth on lipid-containing media. Representative members of other yeast genera found on humans failed to produce these compounds. Addition of lecithin, oleic acids, triolein, or human sebum to the culture media stimulated gamma-lactone production by Pityrosporum species. All yeasts tested produced isopentanol and phenylethanol. Production of gamma-lactones may serve as a valuable characteristic in the identification of organisms of the genus Pityrosporum.     

Upper airway neuromuscular compensation during sleep is defective in obstructive sleep apnea

Obstructive sleep apnea is the result of repeated episodes of upper airway obstruction during sleep. Recent evidence indicates that alterations in upper airway anatomy and disturbances in neuromuscular control both play a role in the pathogenesis of obstructive sleep apnea. We hypothesized that subjects without sleep apnea are more capable of mounting vigorous neuromuscular responses to upper airway obstruction than subjects with sleep apnea. To address this hypothesis we lowered nasal pressure to induce upper airway obstruction to the verge of periodic obstructive hypopneas (cycling threshold). Ten patients with obstructive sleep apnea and nine weight-, age-, and sex-matched controls were studied during sleep. Responses in genioglossal electromyography (EMG(GG)) activity (tonic, peak phasic, and phasic EMG(GG)), maximal inspiratory airflow (V(I)max), and pharyngeal transmural pressure (P(TM)) were assessed during similar degrees of sustained conditions of upper airway obstruction and compared with those obtained at a similar nasal pressure under transient conditions. Control compared with sleep apnea subjects demonstrated greater EMG(GG), V(I)max, and P(TM) responses at comparable levels of mechanical and ventilatory stimuli at the cycling threshold, during sustained compared with transient periods of upper airway obstruction. Furthermore, the increases in EMG(GG) activity in control compared with sleep apnea subjects were observed in the tonic but not the phasic component of the EMG response. We conclude that sustained periods of upper airway obstruction induce greater increases in tonic EMG(GG), V(I)max, and P(TM) in control subjects. Our findings suggest that neuromuscular responses protect individuals without sleep apnea from developing upper airway obstruction during sleep.     

High-performance capillary electrophoresis: Protein charge estimations and peptide mapping by complexation electrophoresis

Two high-performance capillary electrophoretic (HPCE) methods are presented: The first methodology provides a procedure for estimating the isoelectric points of proteins in the absence of chaotropic agents with charge reversal Micro-Coat capillaries. The second method provides an optimized peptide mapping methodology for protein characterization that employs ion-pairing reagents to optimize the HPCE separation. Advantages and limitations of each methodology are discussed in terms of theory and practical experience. Both methodologies are applicable to a variety of proteins and both enhance our ability to characterize proteins on a molecular level.