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)     

Interaction of extracellular Pseudomonas lipase with alginate and its potential use in biotechnology

Summary Extracellular Pseudomonas lipase is able to interact directly or indirectly with alginate as deduced from the following results: (i) During adsorption chromatography of exolipase the enzyme adsorbed quantitatively to glass beads in the absence of alginate, but not after its preincubation in the presence of the polysaccharide; pretreatment of glass beads with alginate did not prevent enzyme adsorption. (ii) In the presence of alginate exolipase was much more resistant to heat inactivation than in its absence. (iii) In the presence of alginate the increase in exolipase activity caused by the non-ionic detergent Triton X-100 was drastically reduced. (iv) Exolipase could be rapidly and almost completely harvested from cell-free culture fluid of P. aeruginosa 5940 by ethanolic coprecipitation with alginate. After dissolving the coprecipitate in detergent-containing buffer exolipase and polysaccharide could be easily separated by ion-exchange chromatography on DEAE-Sephadex A-25. The coprecipitation method was also successfully applied to exolipases produced by Pseudomonas sp., Chromobacierium viscosum and Rhizopus delamar, thus suggesting potential use of this method in biotechnology.     

Migration and proliferation of primordial germ cells in the rat

Information about early primordial germ cell (PGC) formation and migration in rats is lacking. In utero developed and in vitro cultivated whole rat embryos were studied on days 10-13 postcoitum (p.c.). The development of the PGCs was investigated in serial sections stained for alkaline phosphatase activity. On postcoital day 10, PGCs were found in the invaginating visceral yolk sac endoderm and at the base of the allantois. At day 11 p.c. PGCs were mostly found in the ventral and lateral gut wall or in the mesenchyme between the gut and the future genital ridges. At day 12 p.c. most of the PGCs (94%) could be localised in the mesenchyme or in the future genital ridges. On postcoital day 13 almost all PGCs had reached the now-well-developed genital ridges. Quantitative measurements showed an increase in the number of PGCs from 84 at day 10 p.c. up to 2,768 at day 13 p.c. Only slight differences were found between in vivo and in vitro embryos with respect to the number of PGCs and their developmental pattern. The in vitro culture of whole rat embryos enables the discrimination between the effects of indirect (maternal) and direct action of PGC-toxic agents.     

Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate

A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.     

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.     

The annual reproductive cycle of the freshwater mussel Dreissena polymorpha Pallas in lakes

Summary The annual development of the gonads of Dreissena polymorpha was studied at three sampling sites in two lakes over 3 and 1 1/2 years, respectively. A resting stage occurred after the last spawning in summer/autumn. Oogenesis (accompanied by multiplying segmentation of the oogonia and early growth processes of its oocytes) restarted in specimens at least 1 year old at low temperatures (below 10° C) during winter and early spring. At one location (Fühlinger See) the onset of the spawning season was correlated with an increase of water temperatures above 12° C. At 2 m depth, two main spawning periods in May and August were normally recognized, the first at temperatures of 12–16° C, the second at 16–21° C. It was clearly demonstrated for the first time in Dreissena polymorpha that the oocytes became mature in successive cohorts within one gonad. A female mussel may spawn several times during the reproductive season. At 9 m depth, the onset of spawning also started at about 12° C; this occurred in late summer, with two spawning periods within 1 month at a temperature range of 12–16° C. At another location (Heider Bergsee) the size of the gonads and the oocytes was reduced during April of both years studied, when food supply was low simultaneously with rapidly rising water temperatures in this shallow lake. There was no spawning period during spring. The major spawning period was delayed until July (temperatures 19–22°C). This shows (1) the synchronizing influence of low winter temperatures on the annual reproductive cycle and (2) a temperature threshold of at least 12° C for the start of the spawning processes. The results are discussed with regard to the geographical limits of further spread of Dreissena polymorpha.