A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na 2COs followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form . Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al . (1974, J. Cell Biol . 61 :213-231) . The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum . In the accompanying paper (1982, J. Cell Biol . 93 :103-110) the procedure is applied to peroxisomes and mitochondria .

Tuma, Pamela L., and Ann L. Hubbard. Transcytosis: Crossing Cellular Barriers. Physiol Rev 83: 871–932, 2003; 10.1152/physrev.00001.2003.—Transcytosis, the vesicular transport of macromolecules from one side of a cell to the other, is a strategy used by multicellular organisms to selectively move material between two environments without altering the unique compositions of those environments. In this review, we summarize our knowledge of the different cell types using transcytosis in vivo, the variety of cargo moved, and the diverse pathways for delivering that cargo. We evaluate in vitro models that are currently being used to study transcytosis. Caveolae-mediated transcytosis by endothelial cells that line the microvasculature and carry circulating plasma proteins to the interstitium is explained in more detail, as is clathrin-mediated transcytosis of IgA by epithelial cells of the digestive tract. The molecular basis of vesicle traffic is discussed, with emphasis on the gaps and uncertainties in our understanding of the molecules and mechanisms that regulate transcytosis. In our view there is still much to be learned about this fundamental process.

No abstract

An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane . 97 % of the incorporated isotope is in the erythrocyte ghost and 3 % is associated with hemoglobin . No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents . A 6 million-fold excess of chloride ions inhibits iodination by no more than 50 % . Incorporation of up to 1 X 10 6 iodide atoms into a single erythrocyte membrane results in no significant cell lysis . The incorporated label is exclusively in tyrosine residues as monoiodotyrosine . 10-157 of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or 125 I . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 X 106 iodide atoms per ghost does not alter this pattern . Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 4070 of the total label . Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60 % of the total label . Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane . Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane . A combination of protease treatment and double labeling with 1251 and 131I does not reveal the appearance of previously unexposed proteins.