Spheroplasts

Figure 2. PemB cellular localisation. (A) Fractionation of E. chrysanthemi cells by spheroplasting. Lane 1, culture supernatant lane 2, total cell lysate lane 3, periplasmic fraction lane 4, crude membrane fraction lane 5, cytoplasmic fraction. (B) Detergent extraction of PemB from E. chrysanthemi A837 cell envelopes. Lane 1 crude envelope fraction lane 2 Triton-soluble fraction lane 3 Triton-insoluble fraction lane 4 Sarkosyl-soluble fraction lane 5 Sarkosyl-insoluble fraction.

Pope, S., Baker, J.M. Parish, J.H. (1984) Assay of cytotoxicity and mutagenicity of alkylating agents by using Neurospora spheroplasts. Mutat. Res., 125, 43-53... 

Release by spheroplasting SE-cellulose chromatography Chromatography on hydroxylapatite Osmotic release... 

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

By forming spheroplasts from normal cells, Torriani (22) showed that pools of monomer but no alkaline phosphatase (active dimers) exist in the endoplasm and concluded that dimerization occurred outside the endoplasm. Fifteen percent of the enzyme exists as monomers associated with particles in the endoplasm that are larger than ribosomes. 

Schlesinger (23) has shown that under conditions permitting protein synthesis spheroplasts are able to produce the subunits that appeared in the medium, but they are not able to dimerize. It appears then that the monomers are transported out of the endoplasm before being dimerized (24, 25). However, the movement of the monomers from the endoplasm to the periplasmic space followed by their dimerization still requires explanation. 

Syntactic foamed plastics (from the Greek ovvxa C, to put together) or spheroplastics are a special kind of gas filled polymeric material. They consist of a polymer matrix, called the binder, and a filler of hollow spherical particles, called microspheres, microcapsules, or microballoons, distributed within the binder. Expoxy and phenolic resins, polyesters, silicones, polyurethanes, and several other polymers and oligomers are used as binders, while the fillers have been made of glass, carbon, metal, ceramics, polymers, and resins. The foamed plastic is formed by the microcapsular method, i.e. the gas-filled particles are inserted into the polymer binder1,2). 

Takeuchi M, Nakajima M, Ogita T, Inukai M, Kodama K. Furuya K, Nagaki H, Haneishi T (1989) Fosfonochlorin, a New Antibiotic with Spheroplast Forming Activity. J Antibiot 42 198... 

Henner.W.D., I. Kleber, and R. Benzinger. 1973. Transfection of Escherichia coli spheroplasts. 3. Facilitation of transfection and stabilization of spheroplasts by different basic polymers./. Virol. 12 741—747. 

This is a procedure increasingly used to introduce DNA into cells and various methods are available. I have experience with the calcium phosphate technique which is described below but other techniques are described by Gorman (1985) and Spandidos and Wilkie (1984). For example, Bethesda Research Ltd. supply liposomes which will mediate the uptake of nucleic acids at high efficiency (Feigner and Ringold, 1989) and with practice and the appropriate apparatus material may be injected directly into cells (Ansorge and Pepperkok, 1988). Electroporation is particularly useful for introducing DNA into plant spheroplasts. 

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