Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Modification of membrane phospholipid composition by choline analogues induces differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia M1 cells could be induced by various inducers to form Fc receptors, phagocytize, produce lysozyme, and change into forms that were morphologically similar to macrophages and granulocytes. Previous experiments showed that change in phospholipid metabolism was associated with cell differentiation. In the present experiment, culture of M1 cells with choline analogs such as N-monomethylethanolamine and N,N′-dimethylethanolamine resulted in accumulation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N′-dimethylethanolamine in the cell membranes. This change upon treatment with choline analogs was associated with morphological and functional differentiation of the M1 cells into macrophages and granulocytes. These results suggest that phospholipid metabolism is involved in the mechanism of differentiation of M1 cells.     

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells.

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducers including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polyacrylamide slab gel electrophoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with L-[14C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells. P180 was solubilized from 125I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Glyphosine,a plant growth regulator,affects chloroplast membrane proteins

Glyphosine (N,N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10^?4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll $\text{a}\text{b}$ apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll $\text{a}\text{b}$ apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes.     

Evidence of far ultraviolet light-mediated changes in plasma membrane structure and function

Irradiation of plasma membrane preparations with 254 nm light increases its apparent microviscosity as measured with fluorescent polarimetry. Doses of $\text{3 · 10}{}^4$ J/m² increase the fluorescent polarization of a diphenylhexatriene probe by 10%. A similar increase is seen when whole cells are irradiated. The fate of membrane protein following irradiation was examined using SDS-polyacrylamide gel electrophoresis. Increasing the 254 nm doses reduces the amount of material in distinct bands on the gel and increases the amount of very low mobility material. No new bands of Coomassie blue staining material were observed. Irradiation of whole cells inhibited their attachment to concanavalin A-coated surfaces, an indication of a change in membrane function.     

Internal and external membrane proteins of the cyanobacterium, Synechococcus cedrorum

The protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Synechococcus cedrorum, were analyzed with the aid of site-specific labels. Using membranes labeled with ³⁵S, about 50 membrane proteins can be detected by sodium dodecyl sulfate acrylamide gel electrophoresis. Approximately half of the proteins are accessible to modification by the impermeant probe, lactoperoxidase, indicating that they have surface-exposed domains. At least six of these external proteins can be removed by EDTA washing; the correspondence in molecular weights between five of these EDTA-extractable proteins and those of typical chloroplast coupling factor preparations may indicate that they are subunits of a membrane-bound ATPase. The photoactive, lipophilic compound, [¹²⁵I]iodonaphthyl azide, was used to label protein domains in contact with the lipid bilayer. Iodonaphthyl azide modification led to a labeling pattern significantly different from that seen with lactoperoxidase. In particular, proteins in the 13 000–20 000 dalton range that were labeled poorly or not at all by lactoperoxidase were heavily modified by iodonaphthyl azide.Photosystem I and II particles, extracted from the membrane by digitonin treatment, were iodinated by lactoperoxidase after isolation. The PS I particles acted as a relatively tight complex, with most of the proteins remaining inaccessible to surface modification. The PS II particles, on the other hand, responded as a more open structure, with most of the subunits yielding to lactoperoxidase iodination. Similar studies on a highly fluorescent, temperature-sensitive mutant of S. cedrorum revealed a different organization of the PS II complex. This mutant, when grown at 40°C, inserts a 51 kdalton polypeptide in place of a 53 kdalton protein. This protein also replaces the 53 kdalton species in the PS II complex of the mutant after 40°C growth. The structure of this complex is altered in that more sites become accessible to lactoperoxidase. This is particularly true of the 51 kdalton protein, which is barely labeled in wild-type PS II complexes.     

Topology of membrane sulfhydryl groups in the human erythrocyte Demonstration of a non-reactive population in intrinsic proteins

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, $\text{N-}\text{ethylmaleimide}$. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations.These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled $\text{N-}\text{ethylmaleimide}$ or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with $\text{N-[ethyl-2-}{}^3\text{H]}\text{ethylmaleimide}$.The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive.After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards $\text{N-}\text{ethylmaleimide}$. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction

Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with $\text{N-}\text{ethylmaleimide}$, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH₂ groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH₂ groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH₂ groups are not involved in the reaction.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeant labeling experiments indicated that approximately 40% of these proteins were exposed on the extracytoplasmic surface of the plasma membranes of these cells. The studies demostrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.     

Histone phosphorylation in phorbol ester stimulated and β-adrenergically stimulated mouse epidermis in vivo and characterization of an epidermal protein phosphorylation system

Under certain physiological conditions a change i n the phosphorylation of histones in mouse epidermis in vivo was observed. Thus a single local application of the tumor-promoting mitogen 12-O-tetradecanoylphorbol-13-acetate caused a long-lasting increase of histone H1 phosphorylation which paralleled stimulated cell proliferation. Injection of the antimitotic β-adrenergic agonist isoproterenol led to a temporatory decrease in the rate of phosphorylation of H1, H2A and H2b immediately after cyclic AMP accumulation. A complete protein phosphorylation system could be demonstrated in mouse epidermis homogenates. The following enzyme activities were partially purified and characterized: a cyclic AMP-dependnet histone kinase; a ‘casein kinase’ and an ‘unsopecific’ protein kinase; a histone-specific protein phosphatase; and two ‘unspecific’ phosphoprotein phosphatases. In addition, a stimulatory effect of cyclic GPM on histone phosphorylation was observed. The enzymes were found to be predominantly localized in the 105 000 × g supernatant, but a small proportion of protein kinase and phosphatase activity could be regularly demonstrated in cell nuclei.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of proteins solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-³²P]ATP. The ³²P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and alkaline phosphatase. Two Ca²⁺-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La³⁺-ions (20 μM) in a similar way as the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na⁺. This phosphoprotein comigrates with a preparation of renal $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. In brush border membrane preparations the Ca²⁺-induced and the Na⁺-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca²⁺-pump and Na⁺-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

RNA metabolism of murine leukemia virus: detection of virus-specific RNA sequences in infected and uninfected cells and identification of virus-specific messenger RNA

Virus-specific RNA sequences were detected in mouse cells infected with murine leukemia virus by hybridization with radioactively labeled DNA complementary to Moloney murine leukemia virus RNA. The DNA was synthesized in vitro using the endogenous virion RNA-dependent DNA polymerase and the DNA product was characterized by size and its ability to protect radioactive viral RNA. Virus-specific RNA sequences were found in two lines of leukemia virus-infected cells (JLS-V11 and SCRF 60A) and also in an uninfected line (JLS-V9). Approximately 0.3% of the cytoplasmic RNA in JLS-VII cells was virus-specific and 0.9% of SCRF 60A cell RNA was virus-specific. JLS-V9 cells contained approximately tenfold less virus-specific RNA than infected JLS-VII cells. Moloney leukemia virus DNA completely annealed to JLS-VII or SCRF 60A RNA but only partial annealing was observed with JLS-V9 RNA. This difference is ascribed to non-homologies between the RNA sequences of Moloney virus and the endogenous virus of JLS-V9 cells.Virus-specific RNA was found to exist in infected cells in three major size classes: 60–70 S RNA, 35 S RNA and 20–30 S RNA. The 60–70 S RNA was apparently primarily at the cell surface, since agents which remove material from the cell surface were effective in removing a majority of the 60–70 S RNA. The 35 S and 20–30 S RNA is relatively unaffected by these procedures. Sub-fractionation of the cytoplasm indicated that approximately 35% of the cytoplasmic virus-specific RNA in infected cells is contained in the membrane-bound material. The membrane-bound virus-specific RNA consists of some residual 60–70 S RNA and 35 S RNA, but very little 20–30 S RNA. Virus-specific messenger RNA was identified in polyribosome gradients of infected cell cytoplasm. Messenger RNA was differentiated from other virus-specific RNAs by the criterion that virus-specific messenger RNA must change in sedimentation rate following polyribosome disaggregation. Two procedures for polyribosome disaggregation were used: treatment with EDTA and in vitro incubation of polyribosomes with puromycin in conditions of high ionic strength. As identified by this criterion, the virus-specific messenger RNA appeared to be mostly 35 S RNA. No function for the 20–30 S was determined.     

Immunoblot detection of pyridoxal phosphate binding proteins in liver and hepatoma cytosolic extracts

A monoclonal antibody, highly selective for the 5'-phosphopyridoxyl group, can be used to detect cytosolic pyridoxal-5'-phosphate binding proteins by an immunoblot procedure. This technique, when applied to sodium borohydride-treated cytosolic extracts obtained from normal rat liver at various stages of development as well as several liver-derived Morris hepatomas, reveals patterns of pyridoxal-5'-phosphate binding proteins that are characteristic of the various sources of cytosol. These findings suggest that there are developmental and tumor-specific requirements for pyridoxal-5'-phosphate, the coenzymatically active form of vitamin B-6.     

Fibrillogenesis of apomyoglobin facilitated by aggregation sequence of yeast Sup35 in various regions

To examine the effect of aggregation sequence QGGYQQQYNP from yeast Sup35 on fibril formation of sperm whale apomyoglobin (apoMb), we constructed several mutants via substitution. Urea-induced unfolding of apoMb confirms that the substitution of the aggregation sequence does not significantly affect the stability of the mutants compared to wild type (WT) at pH 4.2. Under this condition, however, despite the difference in rate most apoMb mutants form fibrils more readily than WT with distinct morphology. These results suggest that the aggregation sequence facilitates fibril assembly of apoMb at acidic pH in vitro and this facilitation depends on the regions replaced.     

Effects of phospholipase A2 treatment of human erythrocyte membranes on the rates of spectrin-actin dissociation

This work examines the extent to which alterations in the composition of the phospholipid bilayer of the erythrocyte membrane influences the stability of the association of the ‘cytoskeletal network’ to the rest of the membrane. Rates of spectrin-actin dissociation at low ionic strength were used as a measure of the stability, and composition of the phospholipid bilayer was altered by the action of the enzyme phospholipase A₂. Hydrolysis of all the phosphatidylcholine of the outer leaflet of the bilayer had no effect on dissociation rates, whether or not the hydrolysis products were extracted with albumin. Hydrolysis of inner leaflet phospholipids increased the rates by up to 2-fold if the hydrolysis products were not extracted; for ?50% hydrolysis, the rates were unaffected if the hydrolysis products were extracted. The moderate magnitudes of the increases in dissociation rates indicate that interactions between the ‘cytoskeletal network’ and the phospholipid bilayer are not a decisive factor in maintaining the stability of the membrane, at least under low ionic strength conditions.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI⁺D⁺, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI⁺D^?, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI⁺D^? compared to the MGI⁺D⁺ clones. The MGI⁺D^? clones produced an unusual polyunsaturated C_20:5 fatty acid at 28°C, whereas the MGI⁺D⁺ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI⁺D^? clones than of the MGI⁺D⁺ clones. The cells of MGI⁺D⁺ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.