Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs-indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase-suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that-in line with the Dynamic Exchange Model-the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Structural and functional roles of non-bilayer lipid phases of chloroplast thylakoid membranes and mitochondrial inner membranes.

The 'standard' fluid-mosaic membrane model can provide a framework for the operation of the photosynthetic and respiratory electron transport systems, the generation of the proton motive force (pmf) and its utilization for ATP synthesis according to the chemiosmotic theory. However, this model, with the bilayer organization of all lipid molecules, assigns no function to non-bilayer lipids - while in recent years it became clear that the two fundamental energy transducing membranes of the biosphere, chloroplast thylakoid membranes (TMs) and inner mitochondrial membranes (IMMs), contain large amounts of non-bilayer (non-lamellar) lipid phases. In this review, we summarize our understanding on the role of non-lamellar phases in TMs and IMMs: (i) We propose that for these membrane vesicles the dynamic exchange model (DEM) provides a more suitable framework than the 'standard' model; DEM complements the 'standard' model by assuming the co-existence of bilayer and non-bilayer phases and their interactions, which contribute to the structural dynamics of the membrane systems and safe-guard the membranes' high protein:lipid ratios. (ii) Non-bilayer phases play pivotal roles in membrane fusion and intermembrane lipid exchanges - essential processes in the self-assembly of these highly folded intricate membranes. (iii) The photoprotective, lipocalin-like lumenal enzyme, violaxanthin de-epoxidase, in its active state requires the presence of non-bilayer lipid phase. (iv) Cardiotoxins, water-soluble polypeptides, induce non-bilayer phases in mitochondria. (v) ATP synthesis, in mammalian heart IMMs, is positively correlated with the amount of non-bilayer packed lipids with restricted mobility. (vi) The hypothesized sub-compartments, due to non-lamellar phases, are proposed to enhance the utilization of pmf and might contribute to the recently documented functional independence of individual cristae within the same mitochondrion. Further research is needed to identify and characterize the structural entities associated with the observed non-bilayer phases; and albeit fundamental questions remain to be elucidated, non-lamellar lipid phases should be considered on a par with the bilayer phase, with which they co-exist in functional TMs and IMMs.

Chemiluminescent detection of oligoclonal immunoglobulins after isoelectric focusing and affinity-mediated immunoblotting.

BACKGROUND AND AIMS: Detection of oligoclonal IgG (o-IgG) in the cerebrospinal fluid (CSF) not found in serum is the principal laboratory test to support a diagnosis of multiple sclerosis. The aim of this study was to compare chemiluminescent and chromogenic detection of oligoclonal immunoglobulins in the cerebrospinal fluid and serum after their separation by means of isoelectric focusing followed by immunoblotting. METHODS: A set of experiments was designed to detect oligoclonal immunoglobulins by means of alkaline phosphatase BCIP/NBT substrate and chemiluminescent peroxidase substrate. RESULTS: Based on visual evaluation of signals, chemiluminescent detection requires about a 4 times lower amount of applied protein than very sensitive BCIP/NBT chromogenic detection. Very good correlation between methods has been shown for oligoclonal IgG. Antigen-specific oligoclonal IgG could be demonstrated by both methods although the pattern was clearer using chemiluminescence. In one patient, oligoclonal IgD bands barely visible by BCIP/NBT were convincingly demonstrated by chemiluminescence. CONCLUSION: Chemiluminescent detection is a feasible option for oligoclonal immunoglobulin detection and could be used in cases when the sensitivity needs to be improved. Further studies and method optimisation are warranted.