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D F Wallach

Publications and source records attributed to D F Wallach.

At least 91 records · Page 5Linked to original sources
PubMed1976

Cooperative binding of concanavalin A to thymocyte membrane vesicles augments uptake of alpha aminoisobutyrate.

(1) Membrane vesicles from rabbit thymocytes accumulate alpha-aminoisobutyrate in the presence of 0.1 M NaCl. Uptake is 1/2 maximal after about 2 min and reaches a plateau value (61 pmoles/mg protein) after 30 min. (2) Up to 25 mug concanavalin A/ml, binding of the lectin describes a sigmoid curve indicative of a cooperative process. (3) At lectin concentrations up to 8 mug/ml, lectin binding enhances the uptake of alpha-aminoisobutyrate (maximally 30%).

Aminoisobutyric Acids↗
PubMed1976

Erythrocyte membranes undergo cooperative, pH-sensitive state transitions in the physiological temperature range: evidence from Raman spectroscopy.

We have examined the Raman scattering from erythrocyte ghosts at 2700 to 3000 cm-1 (CH-stretching region). Plots of the intensity (I) of the 2930 cm-1 band relative to the intensity of the thermally stable 2850 cm-1 band, i.e., the [I2930/I2850] ratio, as a function of temperature reveal a sharp discontinuity, which at pH 7.4 has a lower limit of 38 degrees and is irreversible above 42 degrees. [I2930/I2850] is stable between pH 7.0 and pH 7.4, but increases or decreases sharply below pH 7.0 or above pH 7.5, respectively. Reduction of pH to 6.5 lowers the transition temperature by about 16 degrees, and a shift to pH 6.0 drops the transition range to 0 to 7 degrees. The above effects of temperature and pH on Raman scattering closely correspond to those detected by studies on the interaction of membrane protein fluorophores and lipid-soluble fluorescence quenchers [Bieri, V. and Wallach, D.F.H. (1975) Biochim. Biophys. Acta 406, 415-423]. Taken together, these results suggest that the transitions represent concerted process, involving hydrophobic amino acid residues and lipid chains at apolar protein-lipid boundaries.

Amino Acids↗
PubMed1976

Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. I. High-yield purification of plasma membrane fragments.

In this first paper of a series comparing the membranes of normal lymphocyte populations from male outbred Syrian hamsters with those of neoplastic transformants (GD 248) induced by simian virus 40, a method is described for the isolation of representative plasma membrane (PM) fragments from both cell types. Multiple criteria were used to monitor the purity and yield of PM material after cell disruption by nitrogen cavitation and after membrane fractionation by a combination of differential centrifugation and isopyknic ultracentrifugation in dextran density gradients. Lactoperoxidase-catalyzed radioiodination before cell disruption was used as an extrinsic surface marker; Na+,K+-activated ATPase, as well as alkaline phosphatase, was used as intrinsic functional PM markers. The distribution of nuclei, mitochondria, lysosomes, and endoplasmic reticulum (ER) during fractionation was monitored by the measurement of DNA, succinate dehydrogenase and monoamine oxidase, beta-glucuronidase and glucose-6-phosphatase, and NADH:lipoamide oxidoreductase, respectively. According to the three PM markers employed, a 15- to 20-fold purification (over homogenate) and a PM yield of about 65% were obtained for both cell categories, with negligible contamination by DNA, mitochondria, lysosomes, and er. The procedure also allowed recovery of 60% of the mitochondria free of other cell elements.

Adenosine Triphosphatases↗
PubMed1976

Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional immune electrophoresis.

The plasma membrane proteins of lymphocyte populations from normal outbred Syrian hamsters were compared with those of a neoplastic transformant line (GD 248) induced by simian virus 40. Both quantitative and qualitative differences were observed. Gradient dodecyl sulfate-polyacrylamide gel electrophoresis revealed 12 major protein components in the membranes of both cell populations. Both membrane categories also contained small amounts of immunoglobulin. Compared with the membranes of the reference cell population, GD 248 membranes showed a 60% decrease of approximately 210,000 daltons of glycoprotein; a 10% reduction of about a 48,000-dalton band and virtually complete loss of a 15,000-dalton component concomitant with a 57% increase in a 52,000-dalton band; fusion to two subcomponents (mol wt approximately 250,000 daltons); and emergence of approximately 120,000 and 30,000 daltons glycoproteins. In addition, the relative mobility of an approximately 95,000-dalton component increased by roughly 0.02 U. Crossed immune electrophoresis in Trition X-100 with heterologous antiserum against GD 248 microsomal membranes revealed both a new component with a high level of electrophoretic mobility and intensification and additional heterogeneity in a strongly antigenic component with a low level of electrophoretic mobility. Crossed-line immune electrophoresis indicated that at least two antigens in the membranes of GD 248 cells lacked the membranes of the reference cell population.

Animals↗
PubMed1975

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.

Antibody Specificity↗
PubMed1975

Variations of lipid-protein interactions in erythrocyte ghosts as a function of temperature and pH in physiological and non-physiological ranges. A study using a paramagnetic quenching of protein fluorescence by nitroxide lipid analogues.

1. Incorporation of stearic acid nitroxides into erythrocyte ghosts markedly depresses the fluorescence of membrane protein tryptophan residues. 5-Nitroxide stearate quenches fluorescence more efficiently than 16-nitroxide stearate. Both compounds exhibit dynamic (diffusion-limited) quenching above 0.28 mumol/mg protein and static quenching at lower nitroxide protein ratios. Static quenching can be attributed to high affinity binding of nitroxide stearates by membrane protein. The dynamic phase represents distribution of the stearate analogues into a fluid lipid system. 2. Protein fluorophores accessible to quenching by a cholesterol analogue, androstane nitroxide, are saturated at low nitroxide/protein ratios (less than 0.14 mumol/mg protein), without resolution of a static quenching phase. This suggests that sterols are segregated away from protein, probably in CLusters". 3. Paramagnetic quenching by stearate nitroxides increases abruptly between 35 and 50 degrees C. This discontinuous enhancement of quenching by temperature is reversible up to 41 degrees C but irreversible at higher temperatures. The discontinuity is also diminished by lowering pH from 7.3 through 6.5 to 6.0. Quenching by androstane nitroxide increases linearly with temperature up to approx. 41 degrees C and then rises exponentially. We attribute the reversible quenching thermotropism detected by stearate derivatives to reversible, thermotropic unfolding and/or depolymerisation of membrane proteins. The irreversible phase, detected also by the sterol derivative can be attributed to non-reversible protein denaturation. 4. Paramagnetic quenching of membrane tryptophan fluorescence by stearate derivatives is minimal at approx. pH 7.1 (35 degrees C) and increases sharply at lower and higher pH values, suggesting that two categories of protein residues, titrating between pH 6 and 8, profoundly influence the association of fatty acyl chains and penetrating protein segments. Quenching by androstane nitroxide exhibits no significant variation between pH 6 and 8, consistent with other data indicating that erythrocyte membrane sterols are segregated from membrane proteins, probably in clusters. 5. Our new approach confirms previous suggestions of a boundary layer of lipid in close association with some proteins in erythrocyte membranes, as well as experiments indicating that the lipid status in this boundary layer depends on that state of membrane proteins. However, sterols appear to be largely excluded from this boundary domain. Our data further show that lipid-protein interactions in erythrocyte membranes can vary significantly with fluctuations of temperature and pH in the physiological range.

Binding Sites↗
PubMed1975

Immunochemical analyses of membrane-bound complement. Detection of the terminal complement complex and its similarity to "intrinsic" erythrocyte membrane proteins.

(1) Membranes of sheep erythrocytes lysed with antibody and human or rabbit complement were solubilized in non-ionic detergents (Triton X-100 or Berol EMU-043) and analysed immunochemically using antisera directed against individual complement components. The precipitation behaviour of membrane-bound C3, C5, C6 and C9 components of complement was examined by immuno-double diffusion, rocket- and crossed immunoelectrophoresis performed in agarose gels containing 1% non-ionic detergent. (2) Membrane-bound C5, C6 and C9 are antigenically altered compared with the native (serum) components. (3) Immuno-double diffusion in the presence of non-ionic detergents reveals formation of C5-C6-C9 complexes on the membranes; these complexes are stable in non-ionic detergent. No complex formation was detected in serum between native C5, C6 and C9 components. There was also no evidence for complexing between membrane-bound C3, C4 or membrane proteins and the "late-reacting" complement components. (4) The extractability of complement components by various manipulations has been studied by use of quantitative rocket immunoelectrophoresis. Up to 65% of membrane-bound C3 is readily extracted by dialysis of membranes against 1mM EDTA, pH 8.0, 100 mM EDTA, pH 8.0, 1.2 NaCl plus or minus EDTA, by extraction in isotonic buffers at 37 degrees C, by heating at 45 degrees C over several hours, or by treating membranes with 1 mM p-chloromercuribenzoate sulfonate. In contrast, less than 6% of the terminal complement complex can be eluted by any of the described methods or combination of methods. (5) Our data suggest that the terminal complement complex associates with membrane "core" components through apolar interactions.

Animals↗
PubMed1975

Anitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L2C cells. In contrast, greater than 5-10(9) molecules/cell of a nitroxide-derivative of androstane, (17 beta-hydroxy-4',4'-dimethylspiro [5 alpha-androstan-3,2'-oxazolidin]-3'-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol roduction by both normal and leukemic cells (maximum within 2 H). At less than 5-10(9) molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.

Acetates↗
PubMed1975

Carotenoids as a Raman-active probes of erythrocyte membrane structure.

1. Erythrocyte ghosts exhibit resonance-enhanced Raman bands at 1530 cm(-1) and 1165 cm(-1) attributable to v(-C=C-) and v(=C-C=), respectively, of the conjugated polyene chains in carotenoids. In lipid extract of ghosts, these resonance-enhanced bands lie at 1527 and 1158 cm(-1). The spectra indicate the presence of membrane-bound beta-carotene. 2. The resonance-enhanced Raman spectrum of beta-carotene in lecithin liposomes is identical to that obtained with hexane or chloroform solutions. 3. Increasing proportions of cholesterol in cholesterol-lecithin liposomes up to a cholesterol: phospholipid molar ratio of 0.8-0.9 drastically decreases the intensity of both resonance-enhanced bands. 4. In ghosts the carotenoid bands respond to membrane perturbations. Trypsinization, lysolecithin treatment and reduction of pH increase the intensities of the 1530 and 1165 cm(-1) bands. In contrast, a decrease in the intensity of both bands follows equilibration of ghosts for 15 min at approx. 50 degrees C or addition of (0.1%) sodium dodecyl sulfate. 5. We suggest that perturbants known to change lipid-protein interactions in erythrocyte membranes modify the microenvironment and/or configuration of the membrane-bound carotenoid.

Binding Sites↗
PubMed1975

Action of halothane on human erythrocytes. Mechanisms of cell lysis and production of sealed ghosts.

1. Suspension of human erythrocytes in halothane-saturated physiological media induces major cell deformation. 2. Release of halothane from erythrocyte suspensions after equilibrium with the anaesthetic produces complete hemolysis. 3. The membrane fragments isolated after halothane release are in the form of biconcave ghosts, impermeable to macromolecules (lactoperoxidase) and small molecules (ATP). 4. The protein composition of the membranes differs from that of ghosts produced by hypotonic lysis in the lack of components previously shown to be adsorbed at low ionic strength. 5. A hypothesis is presented explaining the action of halothane in terms of both its action on membranes and its capacity to modify water structure. 6. Halothane-induced hemolysis constitutes a simple method for the large-scale production of hemoglobin-depleted, sealed erythrocyte ghosts under physiological ionic conditions.

Adenosine Triphosphatases↗
PubMed1975

Two-dimensional separation of erythrocyte membrane proteins.

1). Erythrocyte membrane proteins eluted with Triton X-100 or dilute EDTA have been separated two-dimensionally by isoelectric focusing in polyacrylamide gels containing 1 percent Triton X-100 plus 8 M urea, followed by electrophoresis using sodium dodecyl sulfate. Characteristic patterns, consistent among 40 healthy donors, were obtained. 2. The resulting patterns contain at least 30 components. The "spectrin" components (sodium dodecyl sulfate Bands 1 and 2) focus in the same pH range. Other membrane components giving single bands in sodium dodecyl sulfate electrophoresis appear to be heterogeneous. 3. Triton X-100, but not EDTA, extracts the principal membrane glycoproteins and the major "intrinsic" protein. Otherwise, proteins preferentially eluted by EDTA extract poorly with Triton X-100 and vice versa. 4. Membrane glycoproteins migrate anodally during electrofocusing and can be purified in a simple, one-step procedure.

Cell Membrane↗
PubMed1975

The structure and thermotropism of thymocyte plasma membranes as revealed by laser-raman spectroscopy.

1. Plasma membranes from rabbit thymocytes have been analyzed by laser-Raman spectroscopy over the 800-3000 cm-1 region and the spectra compared with those of endoplasmic reticulum, as well as relevant liposome systems. 2. Evaluation of the Amide I and Amide III regions indicates that thymocyte plasma membranes, but not endoplasmic reticulum, contain appreciable beta-structure peptide. This conclusion is supported by infrared spectroscopy. 3. Evaluation of the 2890 cm-1: 2850 cm-1 intensity ratio of plasma membranes as a function of temperature, using an integration technique, demonstrates a thermotropic lipid transition centered near 23 degrees C. This transition is less sharp than one observed with egg lecithin in this temperature range. 4. The significance of the thermotropic transition is evaluated in view of the lack of thermotropic lipid-protein segregation detectable by freeze-fracture electron microscopy (Wunderlich, F., Wallach, D.F.H., Speth, V. and Fischer, H. (1973) Biochim. Biophys. Acta 373, 34-43).

Animals↗
PubMed1975

Study of thymidylate synthetase-function by laser Raman spectroscopy.

The Laser-Raman spectra of thymidylate synthetase have been obtained with 488 nm excitation from an argon ion laser. Raman bands observed in the range 600-800 cm-minus-1 have been assigned to functional groups of constituent amino acids. The band positions and intensities in the Amide I (1600-1700 cm-minus-1) and Amide III (1200-1300 cm-minus-1) regions, suggest that the enzyme is a mixture of alpha-helical and unordered conformations. Low levels of beta-structure cannot be excluded. The spectra of the ternary complex formed by reacting thymidylate synthetase with (+)-L-methylenetetrahydrofolate and fluorodeoxyuridylate reveals a new band at 1618 cm-minus-1 assigned to the C=N stretching vibration. This band may be due to formation of dihydrofolate or an iminium ion. The overall secondary structure of thymidylate synthetase does not change on formation of the ternary complex. However, the spectrum of the complex indicates local changes in groups such as ionized carboxyl (1400 cm-minus-1), tryptophan (1003 cm-minus-1) and CH-3, CH-2 deformation modes (1440-1470 cm-minus-1).

Binding Sites↗
PubMed1975

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.

Androstanes↗
PubMed1975

Rapid preparative isolation of major erythrocyte membrane proteins using polyacrylamide gel electrophoresis in sodium dodecylsulfate.

1. We describe a simple method for preparative, sodium dodecylsulfate/polyacrylamide gel electrophoresis of the major proteins in human erythrocyte membranes. 2. The method is based on extraction of prestained proteins from gel slabs. Three different fluorescent dyes (o-phthalaldehyde, fluorescamine and 1-dimethyl-aminonaphthalene-5-sulfonylchloride) have been used for pretaining. The method allows separation of up to 75 mg membrane protein and isolation of mg quantities of all major erythrocyte ghost proteins, while preserving the high resolution of analytical polyacrylamide gel electrophoresis. 3. Yield depends on extraction conditions and the molecular weight of the proteins being eluted. It ranges from 43-48% for protein 1 (apparent mol. wt approx. 310000) and 72-78% for protein 3(apparent mol. wt 87 000-93 000) to 87-93% for protein 6 (apparent mol. wt 35 000). 4. The labile behaviour of the high molecular "spectrin" bands (bands 1 and 2) is described. Extraction at room temperature tends to split these proteins into products of lower molecular weight. In contrast, the minor protein components 2.1 and 2.2 tend to aggregate yielding components 1 and 2. 5. N-terminal amino acid analyses have been performed on proteins 1, 2, 3, 4A, 4B, 5 and 6. Each of these bands contains several N-terminals, most of which appear constant. Some additional N-terminal amino acids vary from one donor to the next.

Amino Acid Sequence↗