Donald F. H. Wallach

Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells

Abstract 1. 1. Plasma membrane and endoplasmic reticulum of free fat cells were isolated in high yield as a mixed microsomal fraction by sucrose gradient centrifugation. 2. 2. The semipermeable behavior of these particles was explored by observing shifts in isopycnic density in continuous polymer (Dextran) gradients over a range of pH and ionic conditions. 3. 3. Using a discontinuous Dextran gradient, a plasma membrane fraction was partially purified. The plasma membrane fraction showed a 12–16-fold enrichment of 5′-nucleotidase over the homogenate. Na + -K + -stimulated ATPase was recovered in highest specific activity in this fraction. Mitochondrial contamination was 3% where endoplasmic reticulum contamination was 27%. 4. 4. A sensitive radioassay for 5′-nucleotidase is described.

Inside-Out Red Cell Membrane Vesicles: Preparation and Puirification

Plasma membranes purified from human red cells were Converted into small vesicleS by disruption in alkaline buffer of low ionic strength. Most of these vesicles were inside-out. The presence of divalent cations prevented this inversion. The inside-out vesicles were separatcd from right-side-out vesicles by centrifugration to equilibrium in dextran density gradients.

[22] Preparation of plasma-membrane fragments from mouse ascites tumor cells

Publisher Summary This chapter discusses the preparation of plasma-membrane fragments from mouse ascites tumor cells. When Ehrlich ascites carcinomas (EAC) cells are disrupted quantitatively under isosmotic conditions by rapid decompression of nitrogen gas, the surface membranes fragment into small vesicles which appear in the “microsomal fraction” on conventional differential centrifugation in 0.25 M sucrose. These plasma membrane fragments are separated from the bulk of other microsomal components through electrostatic and density alterations brought about by divalent cations. Density gradients employing the sucrose polymer Ficoll are used to effect the separation. The distributions of surface antigens and of Na + -K + -dependent adenosine triphosphatase are used to monitor the fate of plasma membrane fragments. Contamination of plasma membrane preparations by endoplasmic reticulum is presently evaluated from their diphosphopyridine nucleotide (DPNH)-diaphorase activity. Only the assays for measuring Na + -K + -dependent adenosine triphosphatase (ATPase), DPNH diaphorase, and “surface antigen” is described. Protein is determined by the ninhydrin procedure. The results are expressed as the increase in rate of ATP hydrolysis which occurs when K + is added to a system already containing Na + .

THE BINDING OF KIDNEY-BEAN PHYTOHEMAGGLUTININ BY EHRLICH ASCITES CARCINOMA.

Abstract 1. 1. The phytohemagglutinin of the red kidney bean agglutinates 3 types of erythrocytes, 4 reticuloendothelial cell types, and 3 mouse tumors with different avidity. Evidence is provided that these cell types have surface receptors in common which are responsible for their agglutination. 2. 2. The reaction of the phytohemagglutinin with Ehrlich ascites carcinoma cells was examined in detail. It was shown that the agglutination of this tumor is not a simple function of phytohemagglutinin binding, but depends upon the state of the cell surface. The affinity constant for the reaction of phytohemagglutinin with Ehrlich ascites carcinoma cells was found to be 1.8·10 7 at 1°. The binding affinity was maximal at 18° and diminished at higher temperatures. The 6.6·10 7 receptor sites per Ehrlich ascites carcinoma cell appeared to be located at the cell surface. 0.3% of the receptor sites had to be occupied before agglutination was observed. The velocity of phytohemagglutinin binding increased with temperature and concentration. The rate constant was observed to diminish as the reaction progressed; thus at 1°, it decreased from 2·10 3 to 0.5·10 3 l·moles −1 ·sec −1 during the first 30 min. 3. 3. The affinity constant for the reaction of phytomegglutinin with human erythrocytes was found to be 8.4·10 7 ·4% of the 5·10 5 binding sites per erythrocyte must be occupied for agglutination to be detectable. Erythrocytes treated with trypsin and neuraminidase showed increased agglutinability.

Separation and Partial Purification of Plasma-Membrane Fragments from Ehrlich Ascites Carcinoma Microsomes

Membrane vesicles arising from the surface of Ehrlich ascites carcinoma cells were separated from the bulk of other microsomal components in Ficoll density gradients. Initial separation depends on the differential action of magnesium ion on various microsomal components. Immunologic and enzymic markers were used to monitor separation and purification steps.

The study of lipid-protein interactions in membranes by fluorescent probes

Abstract 1. 1. We report the fluorescence of 1-anilinonaphthalene-8-sulfonate from 220 mμ up in various solvents, in association with lecithin and lysolecithin and bound to membranes of human erythrocytes. 2. 2. The membrane spectra indicate that there is transfer of electronic excitation energy from some of the tryptophans of membrane-bound 1-anilinonaphthalene-8-sulfonate. 3. 3. Our findings raise the possibility that some or all of the membrane-bound 1-anilinonaphthalene-8-sulfonate is associated with membrane proteins; the dye can therefore not be assumed to act solely as a probe for possible lipid regions in membranes.

The optical activity of plasma membranes and its modification by lysolecithin, phospholipase A and phospholipase C

Abstract 1. 1. The circular dichoroism and optical rotatory dispersion of the plasma membranes of human erythrocytes and of Ehrlich ascites carcinoma cells for the spectral range 190–250 mμ are reported. 2. 2. The spectra indicate the presence of some peptide in an α-helical conformation but also suggest that the helical π °- π − transitions of membrane proteins are of a lower rotational strength and are located at a longer wavelength than the corresponding bands in synthetic polypeptides. This view is supported by the analysis of the spectra by curve-fitting using the Gauss nonlinear least-squares method. 3. 3. The helical n - π − transitions of these membranes have the same spectral position as those of synthetic α-helical polypeptides but are of low intensity and of a arge bandwidth, of which the latter accounts for the red displacement of the optical rotatory dispersion through observed in many membranes. 4. 4. Except for the width of the n - α − bands, the optical activity parameters of these membranes differ from those of synthetic polyptide standards in the same manner, although more so, as do certain globular proteins of which the structure is known from X-ray analysis. These deviations are attributed to the presence of short helices, distorted helices and location of some helical peptide chromophores in a highly polarizable apolar environment. 5. 5. We present the effects upon the membrane optical activity of phospholipase A. phospholipase C, lysolecithin and digitonin and conclude that the protein architecture of these membranes depends upon lipid-protein interactions and/or protein-protein interactions which are sensitive to lipid.

A model for the behavior of vesicles in density gradients: Implications for fractionation

Abstract The need for high resolution in the fractionation of membranous vesicles is addressed in terms of a simple theoretical model. We argue that the equilibrium buoyant density of a semipermeable vesicle reflects an osmotic balance between the shrinking forces exerted by non-penetrating gradient solutes and expanding forces engendered by the excess of Donnan ions associated with charges fixed within the vesicle. The mathematical formulation derived from these assumptions leads directly to explicit predictions of density gradient design optimal for separating vesicle species according to their fixed charge content. Briefly, resolution is favored in gradients whose osmotic and ionic activities are minimal, under conditions which also minimize the fixed charge on the membrane. Available experimental data affirm this hypothesis.

Infrared spectra of plasma membrane and endoplasmic reticulum of Ehrlich ascites carcinoma

Abstract The infrared spectra of dried films of plasma membrane and endoplasmic reticulum are dominated by the Amide I and II bands of membrane proteins and by the OH, CH, CO, PO, COC and POC vibrations of membrane lipids. The two membrane types differ in their lipid—protein associations. High amidation of the proteins of both membranes is indicated by the pH-dependent spectral changes and also by amino acid analysis. Exposure of the membranes to acid pH produces spectral evidence for rearrangement of some membrane peptide into β-conformation.

Protein conformational transitions in the erythrocyte membrane

Abstract Erythrocyte membranes prepared from human red blood cells by hypotonic lysis, contain protein in a mixture of α helical and “unordered” conformations. Using infrared absorption spectroscopy we have demonstrated that in the presence of adenosine triphosphate and Mg 2+ , the protein in these membranes undergoes a transition to an anti-parallel β structure, which is enhanced by the addition of Na + and K + . This transition can be prevented or reversed under conditions which inhibit the adenosine triphosphatase reaction. The results are discussed in the light of data previously obtained by infrared spectroscopy on rat liver mitochondria, in terms of a generalized membrane-energy transduction concept.