TonB protein and energy transduction between membranes

TonB protein couples cytoplasmic membrane electrochemical potential to active transport of iron-siderophore complexes and vitamin B12 through high-affinity outer membrane receptors of Gram-negative bacteria. The mechanism of energy transduction remains to be determined, but important concepts have already begun to emerge. Consistent with its function, TonB is anchored in the cytoplasmic membrane by its uncleaved amino terminus while largely occupying the periplasm. Both the connection to the cytoplasmic membrane and the amino acid sequences of the anchor are essential for activity. TonB directly associates with a number of envelope proteins, among them the outer membrane receptors and cytoplasmic membrane protein ExbB. ExbB and TonB interact through their respective transmembrane domains. ExbB is proposed to recycle TonB to an active conformation following energy transduction to the outer membrane. TonB most likely associates with the outer membrane receptors through its carboxy terminus, which is required for function. In contrast, the novel prolinerich region of TonB can be deleted without affecting function. A model that incorporates this information, as well as tempered speculation, is presented.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

An Inhibitor of the UDP-N-Acetylgalactosamine:GM3, N-Acetylgalactosaminyltransferase: Purification and Properties, and Preparation of an Antibody to This Inhibitor

An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. alpha-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.     

Amphomycin: A tool to study protein N-glycosylation

Radio-labelled amphomycin (³H-amphomycin) forms a complex with dolichylmonophosphate in presence of Ca²⁺. Complex formation has also been documented with retinylmonophosphate and perhydromonoeneretinylmonophosphate. Analysis of the space-filling model suggested both fatty acylated aspartic acid residue at the N-terminus of the lipopeptide and phosphate head group of dolichylmonophosphate are necessary for the complex formation. The binding ability of amphomycin is then utilized to localize dolichylmonophosphate in the microsomal membrane. Studies with microsomal membranes from hen oviduct suggested that dolichylmonophosphate is located in the cytoplasmic side of the membrane.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Regulation of seed number and female incitation of mate competition by a pH-dependent proteinaceous inhibitor of pollen grain germination in Leucaena leucocephala

Summary Leucaena leucocephala generally produces pods with more than 7–9 seeds. This is regulated by the stigmatic inhibition of pollen grain germination when the pollen grains are less than a critical number in the stigma. This number-dependent inhibition of pollen grain germination is effected by a pH-dependent proteinaceous inhibitor active at the stigmatic pH. Only when the pollen grains in the stigma exceed the critical number, they inactivate the inhibitor by collectively raising the stigmatic pH and thus overcoming the inhibition. The adaptive significance of such pre-fertilization mechanism for the female in inciting mate competition among the pollen grains is discussed. The evolution of en masse pollen grain dispersal units is explained as a sexual selection strategy by males in response to such stigmatic inhibition by females.     

Effect of nitrogen form on the growth and nitrate concentration of lettuce

Summary A pot experiment with lettuce involving three N forms each at six application levels, showed that lettuce can be grown satisfactorily with a very low nitrate content when supplied with ammonium sulphate and a nitrification inhibitor. For plants growing on nitrate N, the optimum midrib sap nitrate concentration as maturity approached was about 1400 mg/1 NO₃-N. Large losses of mineral N were observed from the peat medium, even in the absence of plants. A relationship is presented which would enable a lettuce grower to estimate whole-shoot nitrate concentration from a quick test of midrib sapi.e. NO₃-N (mg/kg in fresh shoot) =0.14×NO₃-N (mg/l in sap). Tipburn was worst at intermediate levels of applied N, and was less serious with pure ammonium nutrition than with nitrate.     

Revision ofVerbesina sect.Pseudomontanoa (Asteraceae)

Verbesina sect.Pseudomontanoa is revised. The last treatment of the group byRobinson & Greenman (1899) recognized 5 species; the present treatment recognizes 12 species, 3 of which (V. breedlovei, V. cronquistii andV. olsenii) are described as new. A key to species, phyletic diagram and distribution maps are provided.