Immunoelectrophoretic heterogeneity and cross-reactions of individual “spectrin” components isolated by preparative sodium dodecylsulfate-polyacrylamide-gel electrophoresis

Bjerrum, Ole J.; Bhakdi, Sucharit; Knüfermann, H.; Bøg-Hansen, T.C.

doi:10.1016/0005-2736(74)90103-5

Cited by 28 publications

(4 citation statements)

References 21 publications

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“…N-Terminal amino acids were either not quantitated or were recovered in a yield exceeding one per polypeptide chain. Immunoelectrophoretic studies (Bjerrum et al, 1974(Bjerrum et al, , 1975 provide stronger evidence, though it should be noted that some of the distinct species originally reported were later found to be artifactual (Bjerrum et al, 1975).…”

Section: Discussionmentioning

confidence: 95%

“…Similar degradation could also occur in the course of preparative procedures, especially if other types of blood cells (which are rich in proteolytic enzymes) are not completely eliminated in the preparation of erythrocyte ghosts. A number of reports purport to show that the two spectrin bands do in fact contain multiple polypeptide chains by the criterion of end group analysis (Kniifermann et al, 1973;Green et al, 1974;Fuller et al, 1974), but they are by no means conclusive. N-Terminal amino acids were either not quantitated or were recovered in a yield exceeding one per polypeptide chain.…”

Section: Discussionmentioning

confidence: 99%

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Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization

Schechter¹,

Sharp²,

Reynolds³

et al. 1976

Biochemistry

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Erythrocyte spectrin, isolated by aqueous extraction of erythrocyte ghosts, may be freed from contaminating membrane lipids and small amounts of other proteins by gel chromatography in 5 or 10 mM deoxycholate. The purified protein, in deoxycholate, is a mixture of monomers and dimers, both highly asymmetric molecules. The hydrodynamic properties of the dimer closely resemble those of muscle myosin, and spectrin and myosin also have similar circular dichroism spectra. The proportion of dimer to monomer in the purified protein varies from one preparation to another, an observation for which there is no simple explanation. In the absence of deoxycholate, spectrin associated beyond the dimer stage, possibly by loose end-to-end aggregation involving hydrophobic forces.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization

Schechter¹,

Sharp²,

Reynolds³

et al. 1976

Biochemistry

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“…The other major antigens of the protoplast surface (no. 4 and 17) do not appear to share the same degree of exposure and may be located closer to the membrane surface.…”

Section: Figmentioning

confidence: 97%

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Owen

Saltón

1977

J Bacteriol

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Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

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“…However, there have been several recent reports (36)(37)(38) that each of the two bands of spectrin (on SDS gels) has several N-terminal amino acids and are immunochemically (39) and isoelectrically (40) heterogeneous. It is possible that spectrin could contain several kinds of peptides, each having a different ATPase activity.…”

Section: Discussionmentioning

confidence: 99%

Calcium and magnesium ATPASES of the spectrin fraction of human erythrocytes

et al. 1975

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Using a rapid method of preparation, spectrin has been isolated from human erythrocytes and its ATPase activity investigated. The ATPase activity with calcium has two distinct components, one with optimal activity when calcium and ATP are of equal concentration (low-Ca-ATPase) and another which is activated above 1 mM CaCl2 and is maximal at 100 mM CaCl2. There is also a Mg-ATPase with maximal activity at 10 mM MgCl2. The high-Ca-ATPase of spectrin, but not the low-Ca-ATPase, is inhibited by magnesium, while the Mg-ATPase is inhibited by Ca in excess of ATP. None of these activities exhibits the calcium-stimulated magnesium-dependent activity characteristic of the red cell calcium pump.

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Immunoelectrophoretic heterogeneity and cross-reactions of individual “spectrin” components isolated by preparative sodium dodecylsulfate-polyacrylamide-gel electrophoresis

Cited by 28 publications

References 21 publications

Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization

Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Calcium and magnesium ATPASES of the spectrin fraction of human erythrocytes

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