Monomeric collagen imaged by cryogenic force microscopy.
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Animals↗
Biomedical subjects
K C Yanagimoto
Publications and source records attributed to K C Yanagimoto.
Measuring changes in membrane thickness by scanning tunneling microscopy.
We investigated the feasibility of using the scanning tunneling microscope (STM) as a morphometric tool to measure the thickness of biomembranes. Planar monolayers of oriented purple membrane (PM) were prepared, nitrogen-dried or freeze-etched, and coated with metal. PM thickness was quantified by STM and transmission electron microscopy. STM calibration and the effect of contamination-mediated surface deformation on measurements of PM thickness were evaluated. The thickness of PM attached to mica and glass and the effect of papain on PM thickness were also examined. The apparent thickness of enzymatically modified PM increased after papain treatment. The mean thickness of both nitrogen-dried PM on mica and freeze-etched PM on glass was 4.6 nm. After papain treatment PM thickness on mica increased to 4.8 nm and on glass to 5.4 nm. These results demonstrate that STM analysis of metal-coated planar membrane monolayers can be used to measure changes in average membrane thickness at sub-nanometer resolution.
Amino Acids↗
Scanning tunneling microscopy of planar biomembranes.
We combined planar membrane monolayer techniques with scanning tunneling microscopy (STM) to measure the thickness of metal-coated purple membrane (PM) isolated from Halobacterium halobium. Although the metal coating precluded obtaining high-resolution lateral information, it facilitated obtaining high-resolution vertical information. For example, the apparent mean thickness of planar PM and variations in thickness of enzyme-treated PM could be detected and quantified at sub-nanometer resolution.
Cell Membrane↗
Transmembrane signaling: tumor promoter distribution.
Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.
Cytosol↗
Topography of protein kinase C substrates analyzed by membrane splitting.
We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [32 P]Pi into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [32P]Pi-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.
Cell Fractionation↗
Effect of membrane splitting on transmembrane polypeptides.
We investigated the effect of membrane splitting on the primary structure of human erythrocyte membrane polypeptides. Monolayers of intact, chemically unmodified cells were freeze-fractured and examined by one-dimensional SDS PAGE. Silver-stained gels revealed all major polypeptides that stain with Coomassie Blue as well as all bands that stain with periodic acid Schiff's reagent. Both nonglycosylated and glycosylated membrane polypeptides could be detected at concentrations of only a few nanograms per band. Membrane splitting had no effect on the position or number of bands. Monolayers of intact erythrocytes that had been enzymatically radioiodinated with lactoperoxidase were examined by electrophoresis, fluorography, and liquid scintillation counting. Radioactivity was quantified before and after monolayer formation and splitting, and at several stages of gel staining, drying, and fluorography. Although overexposed fluorographs revealed several minor radioiodinated bands in addition to band 3 and the glycophorins, no new bands were detected in split membrane samples derived from intact cells. These observations support the conclusion that neither the band 3 anion channel nor the glycophorin sialoglycoproteins are fragmented during freeze-fracturing. Although both band 3 and glycophorin partition to the cytoplasmic side of the membrane, preliminary quantitative observations suggest an enrichment of glycophorin in the split extracellular "half" membrane. We conclude that the process of membrane splitting by planar monolayer freeze-fracture does not cleave the covalent polypeptide backbone of any erythrocyte membrane protein, peripheral or integral.
Anion Exchange Protein 1, Erythrocyte↗