Separation of plant membranes by electromigration techniques

The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

On the structure and chromosome location of the 72- and 92-kDa human type IV collagenase genes.

The 72- and 92-kDa type IV collagenases are members of a group of secreted zinc metalloproteases. Two members of this family, collagenase and stromelysin, have previously been localized to the long arm of chromosome 11. Here we assign both of the two type IV collagenase genes to human chromosome 16. By sequencing, the 72-kDa gene is shown to consist of 13 exons, 3 more than have been reported for the other members of this gene family. The extra exons encode the amino acids of the fibronectin-like domain which has so far been found in only the 72- and 92-kDa type IV collagenase. The evolutionary relationship among the members of this gene family is discussed.     

Cell adhesion molecule in the hexactinellid Aphrocallistes vastus

Abstract. The Hexactinellida sponge Aphrocallistes vastus contains a soluble aggregation factor (AF) whose purification has been described in this communication. It is characterized by a S°_20.w value of 37 and a buoyant density of 1.45 g/cm³. The AF is a glycoporteinaceous particle composed of three major protein species; no core structure could be visualized. In the presence of Ca²⁺, the AF causes secondary aggregation of single cells. The aggregation process is temperature, pH, and ionic strength independent within a broad range. Evidence is presented indicating that two (or more) AF molecules are required for the establishment of a stable cell: cell interaction. In contrast to the AFs from demosponges, the hexactinellid AF functions species-unspecifically.     

Unexpected metabolites produced from clomethiazole

The heterocyclic drug clomethiazole is metabolized by body passage partly to small aliphatic molecules. The occurrence of such unexpected metabolites is usually overlooked. An appropriate method for their detection is the comparison of urine profiles during drug intake and after withdrawal of the drug.     

Spatial productivity variations during formation of sapropels S5 and S6 in the Mediterranean Sea: evidence from Ba contents

We investigated five time-equivalent core sections (180-110 kyr BP) from the Balearic Sea (Menorca Rise), the easternmost Levantine Basin and southwest, south, and southeast of Crete to reconstruct spatial patterns of productivity during deposition of sapropels S5 and S6 in the Mediterranean Sea. Our indicators are Ba, total organic carbon and carbonate contents. We found no indications of Ba remobilization within the investigated core intervals, and used the accumulation rate of biogenic Ba to compute paleoproductivity. Maximum surface water productivity (up to 350 g C/m²/yr) was found during deposition of S5 (isotope stage 5e) but pronounced spatial variability is evident. Coeval sediment intervals in the Balearic Sea show very little productivity change, suggesting that chemical and biological environments in the eastern and western Mediterranean basins were decoupled in this interval. We interpret the spatial variability as the result of two different modes of nutrient delivery to the photic zone: river-derived nutrient input and shoaling of the pycnocline/nutricline to the photic zone. The productivity increase during the formation of S6 was moderate compared to S5 and had a less marked spatial variability within the study area of the eastern Mediterranean Sea. Given that S6 formed during a glacial interval, glacial boundary conditions such as high wind stress and/or cooler surface water temperatures apparently favored lateral and vertical mixing and prevented the development of the spatial gradients within the Eastern Mediterranean Sea (EMS) observed for S5. A non-sapropel sediment interval with elevated Ba content and depleted ¹⁸O/¹⁶O ratios in planktonic foraminifer calcite was detected between S6 and S5 that corresponds to the weak northern hemisphere insolation maximum at 150 kyr. At this time, productivity apparently increased up to five times over surrounding intervals, but abundant benthic fauna show that the deep water remained oxic. Following our interpretation, the interval denotes a failed sapropel, when a weaker monsoon did not force the EMS into permanent stratification. The comparison of interglacial and glacial sapropels illustrates the relevance of climatic boundary conditions in the northern catchment in determining the facies and spatial variability of sapropels within the EMS.     

Transfer and expression of the tetracyclin resistance transposon Tn925 in Acetobacterium woodii

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.     

Platelet tropomyosin binding protein is identical with serum albumin

We have shown that the platelet tropomyosin binding protein described in the accompanying paper (Gerhard, M. D., DiGirolamo, P. M., and Hitchcock-DeGregori, S. E. (1985) J. Biol. Chem. 260, 3221-3227) is identical with human serum albumin. The immunological determinants are completely shared; the tryptic peptide maps are the same; the proteins comigrate on two-dimensional gels; and the amino acid sequences of the first 33 amino acids are the same. Although human serum albumin in plasma or commercially prepared protein will not bind tropomyosin-Affi-Gel 15, it will bind following purification from plasma by chromatography on hydroxylapatite.