Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autocatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.     

Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.     

Differential inhibition of glutamine and gamma-glutamylcysteine synthetases by alpha-alkyl analogs of methionine sulfoximine that induce convulsions.

The alpha-methyl and alpha-ethyl analogs of methionine sulfoximine, like methionine sulfoximine, induce convulsions in mice and inhibit glutamine synthetase irreversibly; alpha-ethylmethionine sulfoximine is approximately 50% as inhibitory as methionine sulfoximine and alpha-methylmethionine sulfoximine. However, whereas alpha-methylmethionine sulfoximine and methionine sulfoximine inhibit gamma-glutamylcysteine synthetase markedly, alpha-ethylmethionine sulfoximine does not, nor does administration of the alpha-ethyl analog produce the decrease in tissue glutathione levels found after giving methionine sulfoximine or its alpha-methyl analog. The findings strongly indicate that methionine sulfoximine-induced convulsions are closely associated with inhibition of glutamine synthetase rather than with inhibition of gamma-glutamylcysteine synthetase. The alpha-alkyl methionine sulfoximine analogs cannot be catabolized via the corresponding alpha-keto or alpha-imino acids, and, like other alpha-substituted amino acids, are probably not metabolized to a significant extent in vivo; this suggests that the amino acid sulfoximine molecules themselves, rather than their metabolites, are directly involved in the induction of convulsions. Possible explanations for the reported lack of correlation between the occurrence of convulsions and the levels of glutamine synthetase activity (and its substrates and product) are considered. The findings suggest that studies on the mechanism of induction of convulsions may be extended significantly and refined in biochemical terms by the use of other structurally modified convulsant molecules.     

Transient state isoelectric focusing: effects of zone load and carrier ampholyte concentration on the kinetics of defocusing and refocusing in sucrose density gradients

The kinetics of focusing, defocusing, and refocusing of l-histidyl-l-tyrosine in a sucrose density gradient have been studied utilizing a special apparatus for repetitive scanning of the isoelectric focusing column, employing uv absorption optics and a digital data acquisition system. Starting from a uniform or triangular concentration profile, the band is “focused” and a nearly linear pH gradient is formed during initial focusing. The electrical field is then abolished and free diffusion occurs. The electrical field is then reapplied (“refocusing”) and the band is allowed to sharpen, presumably reapproaching a steady state. The band width was measured quantitatively as the second moment about the mean (square of the standard deviation, σ²). In theory, measurements of σ² versus time permit the estimation of the apparent diffusion coefficient (D) and the isoelectric focusing parameter (pE). If the electrical field strength E and the pH gradient, $\text{d(}\text{pH}\text{)}\text{dx}$, were also measured, then one could calculate the slope of the pH mobility curve of the protein $\text{dM}\text{d(}\text{pH}\text{)}$ evaluated at the isoelectric point. D can be measured during the defocusing stage, and $\text{pE,}\text{D}\text{pE}\text{,}\text{or}\text{D}$ can be measured during focusing or refocusing. Several limitations and difficulties in the verification of this theory have been encountered: First, the apparent diffusion coefficient depends on zone load in approximately a linear fashion. Accordingly, it is necessary to measure D at several zone loads, and then extrapolate to zero load by linear regression techniques. Second, the ampholyte concentration has a marked effect on both D and pE. Here we have no a priori reason to extrapolate to zero ampholyte concentration. Also, at present we have no satisfactory method for measurement of E. These preliminary studies should be helpful in indicating further directions for experimental refinement and for generalization of theory.     

Do pollen beetles need pollen? The effect of pollen on oviposition,survival, and development of a flower‐feeding herbivore

Abstract.  1. Pollen is considered to be an important dietary component for many species of flower-feeding herbivores. Its influence on oviposition site selection by the pollen beetle Meligethes aeneus , and on the development of its larvae was investigated.
2. The effects of pollen presence and absence on adult, egg, and larval incidence in the field, and on larval development in the laboratory were compared through the use of Synergy, a composite hybrid oilseed rape Brassica napus variety comprising male-fertile (with pollen) and male-sterile (without pollen) plants.
3. In the field, adult females were more abundant on male-fertile plants during flowering, and a greater proportion of male-fertile than male-sterile buds were accepted for oviposition. These data indicate a possible role of pollen in oviposition site selection by female pollen beetles.
4. The numbers of first instar larvae on the two plant lines did not differ; however, more second instars were found on male-fertile than on male-sterile flowers. This suggests a greater larval survival on male-fertile plants, possibly due to the more readily available food resources and better nutrition afforded by the presence of pollen.
5. Laboratory experiments confirmed that a diet which included pollen improved survival to adulthood and resulted in heavier pupae and adults; however, pollen was not obligatory for larval survival and development.
6. The pollen beetle, previously thought to be an obligate pollen feeder, is therefore more generalist in its requirements for development. These findings may relate to the nutritional and behavioural ecology of other flower-feeding herbivores.     

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2ⁿ cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.