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Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed
Abstract:
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.
Insights
This study mapped Micrococcus lysodeikticus membrane and cytoplasmic antigens using crossed immunoelectrophoresis. Researchers identified enzymes and surface components, revealing asymmetric membrane structure and aiding cellular fraction analysis.
Area of Science:
- Microbiology and immunology
- Cellular and molecular biology
- Biochemistry
Background:
- Understanding the complex composition and organization of bacterial plasma membranes is crucial for deciphering cellular functions.
- Micrococcus lysodeikticus serves as a model organism for studying bacterial cell envelope structures and antigenicity.
Purpose of the Study:
- To identify and characterize antigens present in the plasma membrane and cytoplasm of Micrococcus lysodeikticus.
- To investigate the asymmetric distribution of antigens within the plasma membrane.
- To establish a method for monitoring antigen distribution and potential cross-contamination in cellular fractions.
Main Methods:
- Crossed immunoelectrophoresis was employed to resolve and identify antigens from Triton X-100-solubilized plasma membranes and cytoplasmic fractions.
- Zymogram staining and lectin binding (immunoaffinoelectrophoresis) were used to identify enzyme activities and carbohydrate residues of specific antigens.
- Absorption experiments with intact protoplasts, isolated membranes, and whole cells were performed to determine antigen localization and accessibility.
Main Results:
- Twenty-seven discrete antigens were identified in the M. lysodeikticus plasma membrane, with 14 accessible only on the cytoplasmic face and 12 on the protoplast surface.
- Specific enzymes like adenosine triphosphatase and succinate dehydrogenase were localized to the cytoplasmic face, while succinylated lipomannan was a major surface antigen.
- Ninety-seven immunoprecipitates were resolved from the cytoplasmic fraction, with catalase, isocitrate dehydrogenase, and polynucleotide phosphorylase identified as unique cytoplasmic components.
Conclusions:
- The study confirmed the asymmetric nature of the M. lysodeikticus plasma membrane, with distinct antigen sets on the cytoplasmic and external surfaces.
- The identified membrane and cytoplasmic antigens provide valuable markers for analyzing cellular fractions and ensuring purity.
- The findings offer a sensitive analytical system for monitoring antigen distribution and cross-contamination in bacterial cell biology studies.