A model for gramicidin A′-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin A723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.Abbreviations NMR nuclear magnetic resonance - DMPC dimyristoylphosphatidylcholine - S molecular order parameter - CSA chemical shift anisotropy - DPPC dipalmitoylphosphati-dylcholine - LPC lysophosphatidylcholine     

Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Allosteric modulation of adenosine receptors

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A₁ receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A₃ allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A₁ and A₃ receptors, whereas limited or no information is available for A_2A and A_2B receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.     

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS)

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations. We showed that R270C substitution strongly reduced β4GalT7 affinity towards xyloside acceptor, thus affecting GAG chains formation. This study establishes the molecular basis for β4GalT7 defects associated with altered GAG synthesis in EDS.     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced 723p3q433717282/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55723p3q433717282/xxlarge948.gif" alt="delta" align="BASELINE" BORDER="0">C. The 723p3q433717282/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of 723p3q433717282/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

High-Sensitivity Mass Spectrometry for Analysis of Posttranslational Modifications

Pulsed fast atom bombardment ionization (pulsed-FAB) mass spectrometry has been developed to improve the sensitivity of tandem mass spectrometry (MS/MS), allowing it to be used for the analysis of very small samples. MS/MS, when used with a magnetic four-sector instrument coupled with the pulsed-FAB system, allows significant enhancement in product ion intensity of over ten-fold in magnitude over conventional FAB. MS/MS was applied to the structural analysis of a unique nuclear protein, designated p28, which was isolated from a histone fraction obtained from starfish testes. The results clearly show that protein p28 is a heterodimer composed of testicular histones H2B and H4 which are cross-linked between Gln⁹ of H2B and Lys⁵ of H4.     

2n-fatty acids from phosphatidylcholine label sphingolipids—A novel role of phospholipase A2?

In order to find out whether there is a phospholipase A₂ (PLA₂)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1-¹⁴C]palmitoyl phosphatidylcholine for 16–18 h or 90 min. After labeling for 16–18 h, ¹⁴C-sphingomyelin (SM), ¹⁴C-ceramide and ¹⁴C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16–18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA₂ (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA₂.     

The role of oxygen in the regulation of the metabolism of aerotolerant spirochetes,a major component of <Emphasis Type="Italic">“Thiodendron”</Emphasis> bacterial sulfur mats

Two spirochete strains isolated earlier from 723h51514x71j/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">Thiodendron723h51514x71j/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> bacterial sulfur mats grew better under microaerobic (0.3–0.5 mg O₂/l) than under anaerobic conditions. The microaerobic growth of these strains was accompanied by a twofold increase in the cell yield and the efficiency of glucose utilization, despite the fact that an additional amount of ATP (and, hence, glucose) was spent in this case for the synthesis of exopolysaccharides. Glucose metabolism under microaerobic conditions gave rise to more oxidized products (acetate and carbon dioxide) than under anaerobic conditions (formate, ethanol, pyruvate, and hydrogen). The paper considers two putative mechanisms implemented by aerotolerant spirochetes: adaptive (the use of a more efficient pathway of glucose catabolism) and protective (an enhanced synthesis of exopolysaccharides and the reduction of hydrogen peroxide by the reduced sulfur compounds thiosulfate and sulfide, yielding elemental sulfur). The formation of 723h51514x71j/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">Thiodendron723h51514x71j/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> bacterial sulfur mats in saltwater environments is also discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 725–733.Original Russian Text Copyright © 2004 by Dubinina, Grabovich, Chernyshova.     

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [¹⁴C]xylose transferred from UDP-d-[¹⁴C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V_max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K_m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [¹⁴C]xylose transfer to silk was alkali labile, and [¹⁴C]xylitol was formed when [¹⁴C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [¹⁴C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [¹⁴C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [¹⁴C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V_max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V_max value close to 20% of that of intact fibroin.