Detergents as tools for the purification and classification of lipid rafts

The relative insolubility of lipid rafts in cold non-ionic detergents is the most widely used method to purify these fascinating membrane domains from intact cells or membranes. Most of what we know about lipid raft function has been derived from experiments utilising detergent insolubility as the basis for raft purification. Recently, a wider range of detergents have been used to purify 'rafts', and rafts have been subclassified based on their differential solubility in different detergents. This minireview critically examines the use of detergents as tools for raft isolation and for the subclassification of rafts.     

Designed triple-helical peptides as tools for collagen biochemistry and matrix engineering

Collagens, characterized by a unique triple-helical structure, are the predominant component of extracellular matrices (ECMs) existing in all multicellular animals. Collagens not only maintain structural integrity of tissues and organs, but also regulate a number of biological events, including cell attachment, migration and differentiation, tissue regeneration and animal development. The specific functions of collagens are generally triggered by specific interactions of collagen-binding molecules (membrane receptors, soluble factors and other ECM components) with certain structures displayed on the collagen triple helices. Thus, synthetic triple-helical peptides that mimic the structure of native collagens have been used to investigate the individual collagen-protein interactions, as well as collagen structure and stability. The first part of this article illustrates the design of various collagen-mimetic peptides and their recent applications in matrix biology. Collagen is also acknowledged as one of the most promising biomaterials in regenerative medicine and tissue engineering. However, the use of animal-derived collagens in human could put the recipients at risks of pathogen transmission or allergic reactions. Hence, the production of safe artificial collagen surrogates is currently of considerable interest. The latter part of this article reviews recent attempts to develop artificial collagens as novel biomaterials.     

Fluorescent sterols as tools in membrane biophysics and cell biology

Cholesterol is an important constituent of cellular membranes playing a fundamental role in many biological processes. This sterol affects membrane permeability, lateral lipid organization, signal transduction and membrane trafficking. Intracellular sterol transport modes and pathways as well as the regulation of sterol metabolism and disposition in various tissues are areas of intense research. Progress is intimately linked to development and use of appropriate analogs, which closely mimic the properties of cholesterol while allowing to be detected by spectroscopic or microscopic methods. This review provides an overview of various fluorescent sterols used in membrane biophysics and cell biology including analogs of cholesterol and cholesteryl esters. Attention is paid to the natural fluorescent sterol dehydroergosterol (DHE). A survey of the many applications of DHE in biological research is presented. Special emphasis is on recent developments in fluorescence microscopy instrumentation to visualize DHE as an intrinsically fluorescent analog of cholesterol in living cells.     

Detergents for the stabilization and crystallization of membrane proteins

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.     

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.     

The use of multiple tools for teaching medical biochemistry

In this work, we describe the use of several strategies employing the philosophies of active learning and problem-based learning (PBL) that may be used to improve the teaching of metabolic biochemistry to medical and nutritional undergraduate students. The main activities are as follows: 1) a seminar/poster system in a mini-congress format (using topics of applied biochemistry); 2) a true/false applied biochemistry exam (written by peer tutors); 3) a 9-h exam on metabolism (based in real publications); 4) the Advanced Biochemistry course (directed to peer tutors, where students learn how to read and criticize real medical papers); 5) experiments about nutrition and metabolism, using students as volunteers, and about free radicals (real science for students); 6) the BioBio blog (taking advantage of the "web age," this enhances out of class exchanges of information between the professor, students, and peer tutors); 7) student lectures on public health issues and metabolic disorders directed to the community and lay people; and 8) the BioBio quiz show. The main objective of these activities is to provide students with a more practical and interesting approach to biochemistry, such as the application of theoretical knowledge to real situations (diseases, experiments, media information, and scientific discoveries). In addition, we emphasize the importance of peer tutor activities for optimized learning of both students and peer tutors, the importance of a closer interaction between students and teaching staff, and the necessity to initiate students precociously in two broad fields of medical activity: "real" basic science and contact with the public (also helping students--future doctors and nutritionists--to be able to communicate with lay people). Most activities were evaluated by the students through written questionnaires and informal conversations, along various semesters, indicating good acceptance and approval of these methods. Good student scores in the biochemistry exams and seminars indicated that these activities are also working as valid educational tools.     

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C₁₂EO₈, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid-water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, ΔG_tl⁰, that reflects the direct detergent-transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent-transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately ΔG_EO⁰ = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.     

Applications of sequence coevolution in membrane protein biochemistry

Recently, protein sequence coevolution analysis has matured into a predictive powerhouse for protein structure and function. Direct methods, which use global statistical models of sequence coevolution, have enabled the prediction of membrane and disordered protein structures, protein complex architectures, and the functional effects of mutations in proteins. The field of membrane protein biochemistry and structural biology has embraced these computational techniques, which provide functional and structural information in an otherwise experimentally-challenging field. Here we review recent applications of protein sequence coevolution analysis to membrane protein structure and function and highlight the promising directions and future obstacles in these fields. We provide insights and guidelines for membrane protein biochemists who wish to apply sequence coevolution analysis to a given experimental system.     

Angiotensin antogonists as pharmacological tools.

The availability of specific competitive antagonists stimulated investigation of the physiological and pathological role of angiotensin (A-II) and permitted the qualitative and quantitative characterization of numerous angiotensin receptor sites. The specific, competitive antogonists for A-II inhibit both the direct actions of A-II on isolated smooth muscle preparations and the stimulation of specific vascular receptor sites by which A-II evokes prostaglandin biosynthesis and release. Converting enzyme inhibitors a) block the action of exogenous A-I; b) lower blood pressure in conditions associated with high plasma renin levels (e.g., two-kidney renal hypertension, dehydrated diabetes insipidus rats, or in hemorrhagic shock); c) enhance responses to exogenous bradykinin (by inhibiting bradykininase); but d) do not block the effects of A-II at its receptor sites. A-II-receptor antagonists a) block the action of both A-I and A-II, b) lower blood pressure in high renin states, but c) have no effect on bradykinin degradation or action. Angiotensin receptor and synthesis antagonists have been shown to decrease the overall peripheral resistance and to reverse the renal cortical vasoconstriction during hemorrhagic shock and to prolong survival time in hemorrhaged dogs. It is our belief that angiotensin antogonists have therapeutic potential in hemorrhagic shock and would be expected (alone or in combination with alpha-andrenergic blockade) to overcome vascular shutdown and enhance organ perfusion (especially in the kidney).     

Genetics and biochemistry of Leishmania membrane transporters

The ability to clone and functionally express genes encoding membrane transporters in Leishmania and related parasitic protozoa has illuminated the processes whereby these parasites acquire nutrients from their hosts. It is now possible to probe the physiological functions of these permeases and investigate their role in drug delivery and resistance.     

Detergents as selective inhibitors and inactivators of enzymes

In order to study the detergent-enzyme interaction and to clarify whether such an interaction produces specific or non-specific effects, we investigated the action of natural and synthetic detergents on enzymatic systems of different levels of complexity (crystalline enzymes, crude homogenates, organ preparations, organisms in toto i.e. rats and germinating seeds). The enzyme-detergent interaction was examined both as a time-independent phenomenon (inhibition) and as a time-dependent phenomenon (inactivation). In in vitro experiments a clear inhibition of pyridine-dependent dehydrogenases by long-chain anionic detergents was found. Cationic detergents have their greatest effect on lipase, LDH, MDH and ICDH from rat liver homogenates. At low concentrations SDS inactivates all the dehydrogenase enzymes studied. With high concentrations (10 mM) of SDS and dodecyltrimethylammonium bromide (C12), there was a sharp and non-specific decrease of enzymatic activities. In the in vivo studies, rats were given detergents to drink; the cationic detergent (C12) was far more effective than SDS with enzymes from both intestine and liver homogenates. SDS and C12 do not seem to interfere with enzyme activities at the beginning of the germination of Pinus pinea and Triticum durum seeds. However a marked reduction of activities does occur at the respective maximum germination times of these seeds. The nonionic detergent is ineffective both as inhibitor and as inactivator.     

Detergents inhibit chloramphenicol acetyl transferase.

Potent inhibition of chloramphenicol acetyl transferase (CAT) by Triton X-100 and Nonidet P-40 was observed. The CAT activity was also moderately inhibited by sodium deoxycholate and sodium dodecyl sulfate, and least by Tween 20. Detergents should, therefore, not be used for cell lysate preparation when CAT activity is used as the reporter in a transient expression experiment.     

Cholesterol depletion and genistein as tools to promote F508delCFTR retention at the plasma membrane.

BACKGROUND/AIMS: F508delCFTR-, but not wtCFTR-, expressing fibroblasts resemble Niemann Pick type C cells in the massive intracellular accumulation of free cholesterol. The recruitment and activation of F508delCFTR by cholesterol depletion was studied. METHODS: Filipin staining, forskolin-stimulated anion efflux and FITC-dextran uptake were studied in control cells and fibroblasts treated with 2-hydroxypropyl beta-cyclodextrin phosphatidylcholine large unilamellar vesicles to deplete cellular free cholesterol. RESULTS: Treatment of F508delCFTR-, but not wtCFTR-, expressing fibroblasts with 2-hydroxypropyl beta-cyclodextrin resulted in a reduction in cellular cholesterol and a potentiation of the forskolin-induced anion efflux. In addition, forskolin also promoted a massive increase in the rate of endocytosis in F508delCFTR fibroblasts, which was absent in genistein- or cyclodextrin-treated cultures. CONCLUSION: The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis.     

Cloned myogenic cells during differentiation: membrane biochemistry and fine structural observations.

Isolated membranes from clones of a permanent line of myogenic cells, L₆, were studied during three temporally consecutive stages of growth: (1) exponentially growing cells, (2) intermediate phase cells, and (3) fused cells. Membrane fractions have been isolated on a sucrose gradient and studied with respect to morphology, enzymatic activity, density, and α-bungarotoxin binding. Changes as a function of differentiation were seen both in the density of the fractions and in the specific activities of several proteins. Membranes characterized by specific enzyme markers generally sediment at higher densities when isolated from exponentially growing cells than when isolated from fused cells. The change in density is especially pronounced for the 5′ nucleotidase, TPNH-dependent cytochrome c reductase, and β-N-acetylglucosaminidase. The specific activity of adenylate cyclase increases in the intermediate phase cells and remains high in the fused cells; that of TPNH-dependent cytochrome c reductase decreases in the intermediate phase cells and remains low in the fused cells. The binding of α-bungarotoxin increases dramatically following fusion. On the other hand, there was little change, less than a factor of two, in the specific activities of a number of enzymes during growth or following fusion. In this group are hexose-6-phosphatase, acid phosphatase, Na⁺,K⁺-activated ATPase, and 5′ nucleotidase. Both our morphological and our biochemical studies suggest that in the exponentially growing cells, the ribosomes are associated largely with membranes, whereas in the fused cells, the ribosomes are largely free.