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Biomedical subjects

D F Wallach

Publications and source records attributed to D F Wallach.

At least 73 records · Page 4Linked to original sources
PubMed1977

Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells.

Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the pI 4.5 and pI 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pI 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively.

Animals↗
PubMed1977

Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40.

Highly purified plasma membranes from hamster lymphocytes transformed by simian virus 40 (GD 248) were compared with the membranes of normal cells by crossed immune electrophoresis, crossed-line immune electrophoresis, and bidimensional isoelectric focusing-immune electrophoresis. Antiserum raised by inoculation of guinea pigs with GD 248 membranes was used as serologic reagent, either directly or after absorption with membranes from normal cells. Bidimensional immune electrophoresis reveals the presence in the plasma membranes of GD 248 cells of at least three antigens not detectable in the membranes from the normal cell population. At least two of these are also present in the mitochondrial membranes of GD 248 cells, but none could be detected in membranes of embryonic fibroblasts. Bidimensional isoelectric focusing-immune electrophoresis indicates that the distinctive antigens of the GD 248 membranes are glycoproteins.

Animals↗
PubMed1977

Regulation of cholesterol biosynthesis by normal and leukemic (L2C) guinea pig lymphocytes.

The cholesterol production of guinea pig leukemic (L2C) lymphocytes preceeds at greater than 30 times the rate found in normal cells. Fatty acid biosynthesis is also enhanced in L2C cells. Exposure of L2C cells to cholesterol/lecithin liposomes does not depress their sterol biosynthesis, in contrast to the behavior of normal lymphocytes [Philippot, J.R., Cooper, A.G. & Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 161-166]. However, 25-hydroxycholesterol, an inhibitor of hydroxymethylglutaryl-CoA reductase (NADPH) [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate limiting enzyme in cholesterogenesis, and 25-hydroxycholecalciferol, a biologically potent form of vitamin D3, block sterol biosynthesis of both normal and L2C lymphocytes [Philippot, j.r., cooper, A.G. & Wallach, D.F.H. (1976) Biochem. Biophys. Res. Commun. 72, 1035-1041]. Moreover, both cell types exchange cholesterol equivalently with cholesterol/lecithin liposomes. The only difference in sterol biosynthesis observed between the two cell types is in the temperature response of the enzyme. Arrhenius plots of this enzyme activity exhibit a prominent discontinuity at about 24 degrees in the case of normal cells, but none in the case of L2C. The activation energies for L2C cells and normal cells, above the normal cell transition temperature, were not significantly different. All of the data suggest that the regulatory defect in L2C lymphocytes arises from a deficiency in these cells' internal membranes.

Androstanes↗
PubMed1977

Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope.

Nucleus- and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells, cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected in membranes of normal lymphocytes [Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977) Proc. Natl. Acad. Sci. USA 74, 643-647]. Indirect immune fluorescence of living, SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 4 degrees produced a distinct cell surface fluorescence; however, above 20 degrees , staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells.

Antigens, Viral↗
PubMed1977

Differences between the structural dynamics of plasma membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40 as revealed by laser Raman spectroscopy.

The Raman spectra of highly purified plasma membranes from SV40-transformed GD248 lymphocytes have been compared with the spectra of the membranes of normal cells over the spectral region 100 cm-1 to 3010 cm-1. Striking differences between the two membrane categories were observed in the thermal response of the CH-stretching and acoustical regions. Analysis of CH-stretching shows that the membranes of normal cells exhibit a thermal transition centered at 7 degrees and approximately 5 degrees wide. The membranes of GD248 cells, in contrast, show a lipid transition centered at -5 degrees and 12-18 degrees wide. Analysis of the acoustical region yields equivalent results. The membrane proteins of normal membranes undergo a large thermotropic transition, starting at 39 degrees (sample temperatures), whereas this transition begins at 23 degrees with GD248 plasma membranes. The results suggest the possibility that SV40-specific membrane proteins may modify the collective thermotropic behavior of both normal membrane proteins and membrane lipids.

Animals↗
PubMed1976

Photoactivated cross-linking of proteins within the erythrocyte membrane core.

We describe the reactions of three lipophilic, photoactivated cross-linking reagents, 1,5-diazidonapthalene, 4,4'-diazidobiphenyl, and the reversible 4,4'-dithiobisphenylazide, with erythrocyte membranes. Cross-linking occurs only upon photoactivation. At pH 7 to 8, only spectrin components are cross-linked by these reagents. At pH 5.0 to 5.5 several additional membrane proteins including the major "integral" membrane proteins are also cross-linked, despite equivalent binding of the cross-linkers at neutral and acid pH. The cross-linking rates of various membrane proteins at pH 5.0 to 5.5 depend distinctly upon duration of photoactivation. Bidimensional electrophoresis of membrane proteins after cross-linking with the reversible cross-linker, 4,4'-dithiobisphenylazide, has allowed for the identification of homopolymeric products of cross-linking (e.g. dimers and tetramers of Band 3) and heterocomplexes (spectrin plus other membrane proteins). The data suggest that at reduced pH, cross-linking can proceed not only at the membrane surface but also in the membrane core.

Azides↗
PubMed1976

Interaction of concanavalin A with rabbit thymocyte plasma membranes. Distinction between low affinity assoication and positively cooperative binding mediated by a specific glycoprotein.

1. We have analyzed the interaction of the mitogenic lectin, concanavalin A, with purified plasma membranes isolated from rabbit thymocytes. 2. Scatchard analyses show that in native membranes binding is positively cooperative at low concanavalin A concentrations and non-interacting at high lectin levels. 3. In contrast, membranes treated with 0.0064 M glutaraldehyde exhibit diphasic Scatchard plots, indicating the presence of high- and low-affinity binding sites. The high-affinity zone corresponds to the region of positive cooperativity in native membranes. 4. The number of high-affinity binding sites per cell-equivalent corresponds approximately to the number of glycoprotein (mol. wt. 55000) molecules (1-10(6)/cell), but account for less than 25% of the total lectin binding. 5. Treatment of membranes with 0.0064 M glutaraldehyde selectively crosslinks the glycoprotein (mol. wt. 55000) and its multimers, correlating directly with the modifications of concanavalin A-binding. 6. We conclude that high-affinity binding of concanavalin A to thymocyte membranes is a cooperative process mediated by the glycoprotein (mol. wt. 55000). We further conclude that the bulk of concanavalin A binding is through low-affinity associations, not involving specific membrane macromolecules.

Animals↗
PubMed1976

Lipid-protein relationships in erythrocyte membranes revealed by paramagnetic quenching of protein fluorescence.

1. Paramagnetic quenching of erythrocyte membrane protein fluorescence by nitroxide-labelled lipid analogues has been studied as a function of temperature and quencher concentration, as well as after cross-linking of membrane proteins by glutaraldehyde. 2. Quenching due to nitroxide stearates reveals a static component, due to binding of quencher molecules to protein, superimposed upon a diffusion-limited component. 3. Static quenching decreases progressively above 35 degree C, a temperature region where a thermotropic discontinuity is known to occur (Bieri, V. G. and Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 415-423). 4. Diffusion-limited quenching becomes progressively more prominent as the temperature is raised above 15 degree C. 5. Exposure of membranes to varying concentrations of glutaraldehyde indicates that membrane proteins relatively poorly accessible to cross-linking are those responsible for the membrane thermotropism above 35 degree C. 6. Protein fluorophores accessible to androstane nitroxide are saturated at a low quencher/protein ratio. This ratio is stable below 35 degree C but increases by 50% between 35 and 55 degree C.

Blood Proteins↗
PubMed1976

Multiple thermotropic state transitions in erythrocyte membranes. A laser-Raman study of the CH-stretching and acoustical regions.

1. We have examined the Raman scattering from erythrocyte ghosts at 2700 cm-1 -3000 cm-1 (CH-stretching region), that at low frequencies due to acoustical vibrations, as well as that due to v (-C=C-) and v (=C-C=) of beta-carotene, in the temperature range of -30 degrees C to +30 degrees C. For comparison we have also evaluated the Rama spectra of liposomes composed of lecithins+/-cholesterol. 2. Plots vs. temperature of the intensities (I) of the approx. 2880 cm-1 and 2932 cm-1 bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the (I2880/I2850) and (I2932/I2850) ratios, revealed discontinuities in both erythrocyte membranes and liposomes. 3. In erythrocyte membranes, plots of (I approximately 2880/I2850) and (I approximately 2932/I 2850) reveal several discontinuities. Those reported by (I approximately 2880/I 2850) occur at +17 degrees C and approx. -4 degrees C. The ratio (I approximately 2932/I 2850) reveals a transition between -20 degrees C and +10 degrees C, 1/2 maximal at -8 degrees C. The ratio (I 1527/I 1158), representing the relative intensity of v (-C=C-) vs. v (=C-C=) of membrane-bound beta-carotene, changes discontinuously with the 17 degrees C transition, but is stable over the rest of the temperature change studied. 4. The data indicate that a major membrane-state transition takes place below 0 degrees C but that an additional transition occurs at approx. 17 degrees C.

Carotenoids↗
PubMed1976

Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone.

Animals↗
PubMed1976

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).

Binding Sites↗
PubMed1976

Altered surface topology and membrane functions of rat thymocytes eluted from nylon wool columns.

(1) Following incubation of thymocytes with nylon wool at 37 degrees C, the eluted cells showed an increase in the number of microvilli per cell and a concominant elongation of the microvilli (0.22 mum versus 1.15 mum. (2) Cyclic adenosine monophosphate (cylic AMP) levels were lowered by 30-50% in nylon wool-treated thymocytes. (3) Nylon wool-treated cells showed an impaired Na+-dependent amino acid transport system (2-aminoisobutyrate) whereas the Na+-independent amino acid transport system (1-aminocyclopentane-1-carboxylate) was unaffected.

Amino Acids↗
PubMed1976

Concanavalin A stimulation modifies the lipid and protein structure or rabbit thymocyte plasma membranes. A laser raman study.

(1) We have compared the laser Raman spectra of isolated plasma membranes from resting rabbit thymocytes and cells mitogenically stimulated with concanavalin A. (2) Major alterations in the CH stretching, CH deformation and CC stretching regions indicate a different lipid architecture in the membranes from activated cells. (3) Spectral changes in the Amide I and II regions, by reference to the spectra of model compounds indicate greater protein amidation in the membranes from stimulated cells.

Animals↗
PubMed1976

Involvement of sulfhydryl groups in the action of the insulin and radiation on thymocyte Na+-dependent amino acid transport.

1,p-Chloromercuribenzene sulfonate concentrations less than 10(-5) M stimulate the uptake by thymocytes of 2-aminoisobutyrate, a non-metabolized amino acid. At concentrations greater than 10(-5) M of this reagent, transport is impaired and cell viability is effected. In contrast, 5,5'-dithiobis-(2-nitrobenzoate) between 10(-4) and 10(-6) M produces only stimulation of 2-aminoisobutyrate uptake after treating for 10 min. 2. Treatment of thymocytes with 10(-4)M 5,5'-dithiobis-(2-nitrobenzoate) reveals at least three categories of reactive SH groups. Titration of the most rapidly reacting category, 4 - 10(7)-7 - 10(7)/cell, activates 2-aminoisobutyrate transport to the same extent as does p-chloromercuribenzene sulfonate. Cells treated with 10(-6) M insulin showed a 30-50% reduction in the number of sulfhydryl groups that could be titrated with 5,5'-dithiobis-(2-nitrobenzoate). In thymocytes treated with 10(-6) M p-chloro(203Hg)mercuribenzene sulfonate, addition of 10(-6) or 10(-9) M insulin before treatment with the sulhydryl reagent again reduces the number of titrable SH groups by 20%. 3. Insulin (10(-10)-10(-6) M) also stimulates 2-aminoisobutyrate uptake, but the effects of insulin and SH blocker are not additive. 4. Insulin, but not p-chloromercuribenzene sulfonate, prevents the impairment of 2-aminoisobutyrate transport caused by gamma-irradiation. Treatment of cells with p-chloromercuribenzene sulfonate prior to irradiation increases the radiation impairment of 2-aminoisobutyrate transport. 5. gamma-irradiation reduces the number of 5,5'-dithiobis-(2-nitrobenzoate) reactive sulfhydryl residues by 37%. 6. A model for the action of insulin and irradiation on 2-aminoisobutyrate transport is presented.

4-Chloromercuribenzenesulfonate↗
PubMed1976

Some physicochemical properties of plasma membrane vesicles.

The plasma membranes of many animal cells can be disrupted into small sealed vesicles that can be purified centrifugally and utilized for studies on membrane transport. The vesicles behave as micro-osmometers. However, the presence of charges fixed at the internal and external surfaces of the membrane walls produce pH levels at these surfaces that deviate considerably from bulk pH. Transverse symmetry of charge distribution further leads to transverse asymmetry of surface pH. Finally, charges fixed at the internal membrane surface produced significant Donnan osmotic effects that depend upon membrane composition and ionic environment.

Animals↗
PubMed1976

Low- and high-affinity concanavalin a binding to thymocyte plasma membrane vesicles.

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (approximately 6 x 106/cell equivalent) low-affinity sites (Ka = 1.3 x 105M-1) anf (b) fewer (approximately 0.4 x 106/cell equipment) specific receptors (Ka = 6.8 x 106 M-1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of approximately 1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concavalin A-binding induces multimerization of the 55,000 D glycoprotein.

Animals↗