The lipoprotein content of the outer-membrane medium vesicles, which are released from Escherichia coli during normal growth, was compared to the lipoprotein content of the corresponding cellular outer membranes.It was found that the medium vesicles contained only 35% free lipoprotein and almost none of the bound lipoprotein when compared with cellular outer membranes. Medium vesicles also had reduced amounts of protein 11* and a protein V ( M , = 16000), while they contained large amounts of pore-forming proteins I and 1amB.A mechanism is proposed in which outer membrane vesicles are formed when the outer membrane expands faster than the underlying peptidoglycan layer. The lack or enrichment of individual proteins in medium vesicles may be determined by their interactions with the peptidoglycan-bound lipoprotein complex.The cell wall of gram-negative bacteria consists of a peptidoglycan layer and an outer membrane. These two layers are linked covalently via the bound form of the outer membrane lipoprotein [I -31.The lipoprotein is the most abundant protein of Escherichiu coli and many other gram-negative bacteria; there are of the order of 40000 such linkages per pm2 of bacterial cell surface, implying that the distance between neighbouring covalent murein-outer membrane connections averages S nm, which is less than a single membrane thickness [I].Despite this intimate and covalent contact between the outer membrane and the peptidoglycan layer, E. coli and other gram-negative bacteria release a significant portion of their outer membrane into the medium in the form of membrane vesicles during normal growth [4-81.Such vesicles have been observed under the electron microscope as outer-membrane 'blebs' that are released from the cell during the initial stages of septation [9,10]. Release of these vesicles is enhanced when protein synthesis is slowed down, for instance by growing a lysine auxotroph under lysine-limiting conditions or by growing E. coli in the presence of chloramphenicol [I 1,121. Adsorption of phage T4 to E. coli also results in a more pronounced release of outer-membrane vesicles into the medium [6,13].Extensive bleb formation has also been observed when a lipoprotein-lacking mutant was grown under conditions of magnesium starvation [14,15], while a mutant lacking the ompA protein a5 well as the lipoprotein showed extensive blebbing even in the presence of normal magnesium concentrations [16]. Comparable observations have been made in Salmonella typhimurium lkyD mutants which have a decreased amount of murein-bound lipoprotein [17-191. We have previously considered the possibility that, given the high average density of covalent lipoprotein-murein linkages in normal E. coli, outer-membrane vesicles may well be derived from envelope areas which are relatively poor in lipoprotein [4] ; the fact that lipoprotein-lacking mutants h z y m e . Lysozyme (EC 3.2.1.17) release considerably more outer membrane material than do normal cells supports this hypothesis.If the lack of (bound) lipoprotein in cert...

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The identification of the free and murein‐bound forms of theEscherichia coli lipoprotein on dodecylsulphate‐polyacrylamide gels was systematically investigated by analyzing the low‐molecular‐weight proteins (Mr < 20000) of both cytoplasmic and outer membranes. The free form of the lipoprotein was identified on 15% polyacrylamide gels as the fastest migrating component (Mr= 7200–7500) of isolated outer membranes; it could be separated from a small cytoplasmic membrane protein (Mr= 6500) which was probably identical to the dicyclohexylcarbodiimide binding proteolipid of the membrane‐bound ATPase. Lysozyme treatment of both outer membranes and murein sacculi failed to convert the murein‐bound lipoprotein into a fragment of uniform size; instead the bound form appeared as a series of bands consisting of lipoprotein bound to one, two,… eight murein subunits. The composition of this ladder depended on the method used to isolate outer membranes. Beside these lipoprotein bands the outer membrane contained two other proteins, III and V; the relation of these proteins to previously described proteins is discussed.

Zinc uptake was studied in synaptosomes, isolated by the Ficoll flotation technique, using the radiotracer 65Zn. True uptake of zinc could be discriminated from binding to the outside of the synaptosomes by the absence of accumulation at 0 degree C and the dependency of the rate of uptake on the medium osmolarity. The zinc uptake, studied in the presence of various zinc-complexing agents, showed saturation kinetics when analyzed in terms of [Zn]free, yielding Km = 0.25 microM. The zinc uptake was independent of both ATP and the Na+ gradient. No efflux of zinc could be demonstrated from preloaded synaptosomes due to the formation of insoluble zinc complexes inside the synaptosomes. The results are discussed in terms of the modulation of diverse neurochemical processes by zinc.

The effect of dietary zinc deficiency on the mossy fiber zinc content of the rat hippocampus was investigated using PIXE (Particle Induced X-Ray Emission) spectroscopy. Using the proton microbeam (60 X 60 microns), 2 mm line-scans were made on hippocampal sections and the data were expressed as absolute zinc concentrations. Values of 55 and 136 ppm (dry weight) were found for the mean background zinc level and the maximum mossy fiber zinc level, respectively, in animals fed a control diet containing 50 ppm zinc. Treatment of these animals with dithizone caused about 50% reduction in the maximum mossy fiber zinc level. Feeding a zinc-deficient diet for 28 days did not cause a decrease in the mossy fiber zinc level, however, feeding the zinc-deficient diet for 90 days reduced the maximum mossy fiber zinc level by about 30%. The results are discussed in relation to the behavioral abnormalities that have been observed in zinc-deficient animals.