Karl-Erik Johansson

Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane.

The membrane proteins from Spiroplasma citri have been resolved into 16 components by SDS-polyacrylamide gel electrophoresis. By this technique it was also shown that the molecular weights of these proteins ranged from 13000 to 160 000. One of the proteins, which had an apparent molecular weight of 26 000 was the most abundant and represented more than 22% of total membrane protein. We have designated this protein spiralin. None of the proteins contained carbohydrate. Spiralin has been isolated by a procedure which involves removal of some membrane proteins with the neutral detergent Tween 20, selective solubilization of the Tween residue in DOC and fractionation of the DOC-soluble material by agarose-suspension electrophoresis. The homogeneity of spiralin was demonstrated by analytical polyacrylamide gel electrophoresis under different conditions and by crossed immunoelectrophoresis. Spiralin appeared to bind less DOC than the other membrane proteins of S. citri. This observation does not imply, however, that the binding of DOC to spiralin is weak. Spiralin was neither soluble in detergent-free buffers nor in Tween 20, which indicated that it is an intrinsic membrane protein. The amino-acid composition of spiralin was quite different from that of the membrane. Spiralin lacked methionine, histidine and tryptophan, and had a low content of glycine, leucine, tyrosine and phenylalanine, but a high content of threonine, alanine and valine.

Selective solubilization with tween 20 of membrane proteins from Acholeplasma laidlawii

Abstract 1. 1. Approx. 70% of the plasma membrane from Acholeplasma laidlawii can be extracted with 2.5% Tween 20. The residue contained a higher proportion of hydrophobic amino acids than the extract. Glucosamine and galactosamine were found only in the extract. The extraction with Tween 20 is unusually selective: polyacrylamide-gel electrophoresis in sodium dodecyl sulfate of the extract and of the residue showed that the extract contained several membrane proteins that were absent from the residue and vice versa . 2. 2. The proteins in the Tween extract were frationated by analytical polyacrylamide-gel electrophoresis in the presence of Tween 20 into three major and two minor zones (see Fig. 6). 3. 3. A laidlawii membranes, completely solubilized in sodium dodecyl sulfate, were submitted to polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Five major and seven minor zones were obtained. This technique also permitted a rough estimate of the molecular weights of the proteins in these twelve zones; values from 15000 to 140000 were obtained. 4. 4. The five protein zones obtained by electrophoresis in Tween 20 (see point 2 above) were further fractionated in sodium dodecyl sulfate by a two-dimensional electrophoresis procedure (see Fig. 7). 5. 5. The yellow color of the A. laidlawii membrane is due to the presence of both flavoprotein(s) and carotenoid material, the latter pigment being either in free form or in association with lipids. 6. 6. An approach to the determination of the geometric localization of components in a membrane is discussed (although not utilized in the present studies). 7. 7. The risk of misinterpretations in membrane studies due to the possible appearance of artificial complexes is discussed.

Localization of the Tween 20-soluble membrane proteins of Acholeplasma laidlawii by crossed immunoelectrophoresis☆

The Tween 20-soluble membrane proteins from Acholeplasma laidlawii have previously been fractionated by preparative agarose-suspension electrophoresis. The fractions obtained have now been characterized by crossed immuno-electrophoresis in the presence of Tween 20 and with antiserum containing antibodies directed against the membrane proteins. This antiserum was also utilized in order to get some information about the location of proteins, i.e. whether they are located on the inside or the outside of the membrane. The method used is based upon crossed immunoelectrophoresis of the Tween 20-soluble membrane proteins as antigens and uses an antiserum that has been depleted of the antibodies that are directed against proteins with antigenic determinants exposed either on the outside of the membrane or on both sides. These two types of antisera (called I and II) can be produced by adding intact cells or washed, lysed cells, respectively, to the original antiserum and then removing the cells with the adsorbed antibodies by centrifugation. If there exists in the intact membrane a protein which has antigenic determinants, e.g. only on the inside of the membrane, a precipitation line corresponding to this protein will appear in crossed immunoelectrophoresis experiments with the original antiserum and antiserum type I, but not with antiserum type II. Using this method we found that probably only one of the Tween 20-soluble proteins is exposed on the outside and three on the inside of the A. laidlawii membrane. These findings, combined with results obtained by digesting and labelling erythrocytes and by immunological investigations of protoplasts of Micrococcus lysodeikticus, may reflect a possible, general feature of the structure of the plasma membrane, namely that most of its proteins are associated with the inner surface of the membrane. There is also some evidence that no protein is buried within the lipid layer, which also has been found for erythrocyte ghosts by a labelling technique, and therefore may be another characteristic architectural feature of plasma membranes.

Neuron-specific enolase in relation to differentiation in human neuroblastoma

The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCl, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.

Solubilization of Spiroplasma citri cell membrane proteins with the anionic detergent sodium lauroyl-sarcosinate (sarkosyl)

1. Up to 90 per cent of the membrane proteins from Spiroplasma citri could be solubilized with the anionic detergent Sarkosyl (sodium lauroyl-sarcosinate). Maximal solubilization was obtained with 6 to 20 mumoles of of detergent per mg of membrane protein. The insoluble residue, comprising about 10 per cent of the membrane protein, contained mainly the protein spiralin, which is quantitatively the major one of this membrane. 2. Mg2+ ions completely prevented solubilization of the membrane proteins at a molar ratio of MgCl2/Sarkosyl greater than 0.5. 3. The selectivity of Sarkosyl was also tested at low detergent concentrations and in the presence of Mg2+ ions. Spiralin was the least soluble protein also under these conditions. Other proteins were not selectively solubilized. 4. An electrophoretical and immunoelectrophoretical approach was used to study the interaction between Sarkosyl and membrane proteins. The results indicated that Sarkosyl should be considered as a mild detergent which usually solubilizes membrane proteins without gross donformational changes. This hypothesis was supported by experiments with a membrane-bound enzyme in the presence of Sarkosyl.

Fractionation of Membrane Proteins from Acholeplasma Laidlawii by Preparative Agarose-Suspension Electrophoresis

A method for preparative purification of two Tween 20-soluble membrane proteins from Acholeplasma laidlawii is described. Their amino acid composition is given. One of the two proteins is a flavoprotein.

Identification of iron- and phosphorus-containing antigens of the Acholeplasma laidlawii cell membrane

Mycoplasmas are prokaryotes, without a cell wall. This property makes them useful for membrane studies, as the only permeability barrier surrounding the cell, is the plasma membrane [l]. Some mycoplasmas have membrane-bound respiratory-associated components [2], but, compared to other prokaryotes, remarkably little is known about how they transport electrons. Most mycoplasmas lack cytochromes as well as ubiquinones [3] and their respiratory system has, accordingly, been characterized as ‘flavin-terminated’ 141. However, iron and acid-labile sulphur have been found in both the membrane and the cytoplasmic fraction of the cells [4]. The NADH oxidase of Acholeplasma laidlawii, which is membrane-bound in this mycoplasma (as well as in the other Acholeplasma species), has been purified after extraction with the neutral detergent Triton X-100 [5]. The enzyme was shown to be a copper-containing iron sulphur flavoprotein composed of 3 peptide subunits. To relate the structure with the function of this enzyme, it is important to establish to which of its subunits iron, copper, and FMN are bound. Incorporation of 59Fe, analysis of membranes by crossed immunoelectrophoresis (CIE), and autoradiography have been used to identify 7 iron-containing membrane protein antigens from Escherichia coli [6]. One of these antigens was identified as the NADH dehydrogenase by a zymogram-staining

Evidence for Protein-Lipid Interactions in Membranes of Acholeplasma Laidlawii

Abstract By extraction of Acholeplasma laidlawii membranes with Tween 20 and sodium deoxycholate a selective solubilization of proteins and lipids can be obtained. Most lipids can be separated from the proteins with certain electrophoretic techniques. Residual lipid composition in different protein fractions indicates that different proteins are surrounded by different lipids.

Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis

Membranes from the wall‐less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS). The lipoglycan has extraction properties, which are similar to those of LPS of gram‐negative bacteria, but it is chemically distinct from bacterial LPS. The membrane‐bound lipoglycan of A. laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes. The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20‐solubilized A. laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6. The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.

Variations in the Antigenic Architecture of the Acholeplasma Laidlawii Cell Membrane

Abstract Immunoabsorption has been used to elucidate the antigenic architecture of the Acholeplasma laidlawii cell membrane. Crossed immunoelectrophoresis was utilized to evaluate the immunoabsorption experiments. The membrane proteins appeared antigenically as endoproteins, non-trans ectoproteins, or ectoproteins. One of the membrane proteins had no antigenic determinants accessible to antibody in the immunoabsorption experiments. Ectoproteins had antigenic determinants expressed to a higher extent on the exterior face of the membrane in the early phase of the growth cycle. When the cells were grown in a medium containing cholesterol and unsaturated fatty acids, the cell membrane became fragile, and immunoabsorption experiments were therefore difficult to interpret.