Solubilization of membranes by detergents

Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes.

As the decade progresses this prediction is rapidly being realized, and microbial enzymes are becoming increasingly important in such diverse fields as medicine, brewing, and timber preservation. The genus Bacillus has played a major role in this development as evidenced by the distribution of the papers read at the Fifth International Fermentation Symposium, 1976 (64). Of 23 papers in the session devoted to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with enzymes from bacilli. Reasons for the predominance of these bacteria in this area of study are several. First, they comprise a group of chemoorganotrophs that can be easily maintained and cultivated and yet are markedly heterogeneous in character. Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic, and acidophilic species are well represented. Furthermore, virtually all 48 species of the genus listed in Bergey's Manual ofDeterminative Bacteriology (92) secrete a variety of soluble extracellular enzymes, which reflects the diversity of the parental habitats. Amylases that can liquefy starch under pressure at 11000 (194) and proteases that are stable and active at pH 12.0 (6) are extreme examples of enzyme adaption. This article will attempt to review the recent literature concerned with the characterization and properties of the exoenzymes synthesized by the bacilli and the control and mechanisms of their synthesis. It is restricted to this genus because the commercial importance of extracellular enzymes and academic interest in the process of sporulation have prompted a considerable amount of research into this general area. Nevertheless, in the final section I have attempted to equate our present knowledge of exoenzyme synthesis in procaryotes other than

Extracellular enzyme synthesis in the genus Bacillus.

Bioactive compounds can be valuable research tools and drug leads, but it is often difficult to identify their mechanism of action or cellular target. Here we investigate the potential for integration of chemical-genetic and genetic interaction data to reveal information about the pathways and targets of inhibitory compounds. Taking advantage of the existing complete set of yeast haploid deletion mutants, we generated drug-hypersensitivity (chemical-genetic) profiles for 12 compounds. In addition to a set of compound-specific interactions, the chemical-genetic profiles identified a large group of genes required for multidrug resistance. In particular, yeast mutants lacking a functional vacuolar H(+)-ATPase show multidrug sensitivity, a phenomenon that may be conserved in mammalian cells. By filtering chemical-genetic profiles for the multidrug-resistant genes and then clustering the compound-specific profiles with a compendium of large-scale genetic interaction profiles, we were able to identify target pathways or proteins. This method thus provides a powerful means for inferring mechanism of action.

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Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways

Publisher Summary This chapter reviews the use of detergents for solubilization of membranes and as a solvent medium for membrane proteins. It summarizes the properties of detergents that have been used for these purposes and presents some facts relevant to the choice of detergents for particular experiments. The lipids and proteins of a native membrane interact with each other in a complex fashion that differs in detail from membrane to membrane. The optimal detergent for a particular membrane or membrane protein has to be found empirically and may depend on the type of experiment one wants to do. It should be noted that exchange of one detergent for another is relatively simple. One may use different detergents for initial solubilization and delipidation for subsequent protein characterization and for reconstitution into a lipid vesicle. Integrity of a protein may require contacts among polar groups that are outside the lipid bilayer in the native membrane, possibly including interactions with peripheral proteins adjacent to the membrane or with nucleic acid in the case of a nuclear or viral membrane. Such contacts are influenced by pH, ionic strength, and specific ions.

J.O. Lampen

Papers

Tunicamycin--an inhibitor of yeast glycoprotein synthesis.

Affinity chromatography purification of penicillinase of Bacillus licheniformis 749/C and its use to measure turnover of the cell bound enzyme

Bacillus licheniformis 749-C plasma membrane penicillinase, a hydrophobic polar protein.

An enzyme which degrades the walls of living yeast.

Evidence for the extrusion of an incompletely folded form of penicillinase during secretion by protoplasts of bacillus licheniformis 749/C1