Publisher Summary This chapter discusses microsomal lipid peroxidation Lipid peroxidation is a complex process known to occur in both plants and animals It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid Lipid peroxidation begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid radical The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes

Microsomal lipid peroxidation.

Calcineurin is a eukaryotic Ca2+- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an act...

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.4.1483

Calcineurin : form and function

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex.

Postsynaptic integration of glutamatergic and dopaminergic signals in the striatum

Publisher Summary This chapter describes the technique for cryofixation. The goal of ultrastructural fixation methods is to preserve the momentary distribution of all components in a system. For molecular systems, this implies the maintenance of the statistical distribution of all solute components in the solvent and the preservation of the size and shape of the macromolecules in the hydrated state. Cryofixation and freeze-storage of biological materials are important problems in many regards: for the storage of living materials, for the storage and processing of proteins, and for the preservation of structural details. The main problem of cryofixation is that the “fixed” structures are thermodynamically unstable at low temperatures. It is important to have several different cryofixation techniques at hand, if the variety of biological materials and the broad spectrum of secondary techniques are considered, whereby each has its requirements.

Cryofixation : A Tool In Biological Ultrastructural Research

Human erythrocytes have been freeze-fractured, and the polypeptides associated with the separate halves of the membrane bilayer have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The transmembrane proteins were differentially separated by the fracture process. Although sialoglycoproteins associated with the outer half of the membrane, the anion transport protein (band 3) mainly remained with the inner half of the membrane. Well-defined fragments of the sialoglycoproteins were produced by the freeze-fracture procedure, indicating that selected covalent bonds of these transmembrane proteins were broken.

Distribution of transmembrane polypeptides in freeze fracture.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia.

Using a radioimmunoassay, we have measured the level of calmodulin- dependent phosphatase (calcineurin) in various subcellular fractions from chick forebrain. Our results revealed high levels of the enzyme in the cytoplasm and microsomes. A considerable amount was also observed in synaptosomes, where it was found exclusively in the synaptoplasm, comprising 0.32% of the total synaptoplasmic protein. Immunocytochemical localization of the phosphatase in isolated synaptosomes supported the biochemical finding. Phosphatase was not detected in nuclei, myelin, synaptic vesicles, and mitochondria. These results suggest that myelin basic protein and histone H1, widely used in biochemical characterization studies of the phosphatase, may not be physiological substrates, and that the cytoplasm, microsomes, and synaptoplasm may prove to be useful sources for the identification of physiological substrates.

https://www.jneurosci.org/content/jneuro/8/4/1245.full.pdf

Quantitative subcellular localization of calmodulin-dependent phosphatase in chick forebrain

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.

Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine

Harold H. Edwards

Papers

Distribution of transmembrane polypeptides in freeze fracture.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia.

Quantitative subcellular localization of calmodulin-dependent phosphatase in chick forebrain

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine