Separation of mammalian cell surface proteins by a two-dimensional gel electrophoresis system

Separation of externally exposed plasma membrane proteins of mammalian cells has been achieved by a new two-dimensional gel electrophoresis system. The proteins were separated in the first dimension on cylindrical polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) and in the second dimension on polyacrylamide slab gels containing 9 M urea, 0.1% SDS, and 0.1% Triton CF10. Using this method we have obtained reproducible high-resolution patterns of cell surface proteins of differentiated rat neuro-tumor cells in culture and of normal rat retinal cells. Different cell types show characteristic cell surface proteins in addition to ubiquitous ones. The number of common surface proteins between two cell types account for approximately half of the total surface proteins. By immunoprecipitation we have also found that rabbit anti-serum against a rat neuronal cell line can recognize most of these external proteins. Since the separation in the first dimension is done in the presence of SDS and the second dimension in the presence of SDS, a non-ionic detergent, and urea, the technique is particularly suitable for proteins that are of poor solubility. In addition to size, net charge and hydrophobicity appear to be important factors in the separation. Virtually all of the proteins that run in the first dimension can be recovered and further separated in the second.     

Effects of thyroid hormone administration on the susceptibility of rat liver chromatin to digestion with micrococcal nuclease

The accessibility of rat liver chromatin to digestion with micrococcal nuclease was investigated in normal, thyroidectomized and thyroid hormone-treated animals. A significant increase in digestibility of chromatin by micrococcal nuclease was produced by thyroid hormone treatment. The DNA in the soluble fraction analyzed by electrophoresis showed identical sizes in thyroidectomized and triiodothyronine-treated animals. However, DNA in the pellet obtained from thyroidectomized animals showed a relatively high concentration of polynucleosomes which were virtually undetectable in the pellet from thyroid hormone-treated animals. Analysis of proteins in the micrococcal nuclease solubilized fraction of chromatin revealed differences between thyroidectomized and thyroid hormone-treated animals. It is suggested that thyroid hormone causes changes in nucleoproteins which alter the structure of chromatin in such a way as to expose more DNA to nuclease attack and/or increases the solubility of released nucleosomes.     

Studies on the oligomeric structure of yeast phosphofructo-kinase by means of cross-linking diimidoesters.

Intracellular cross-linking of yeast phosphofructokinase with a series of diimidoesters of different chain length resulted in the appearance of tetramers as largest cross-linked product of the enzyme subunits. The native enzyme is evidently composed of eight subunits being arranged in two tetramers α₄β₄. In the tetramers the monomers are probably assembled in tetrahedral geometry.     

Azidophenantridinium compounds as photoaffinity labels of cholinergic proteins

The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these compounds as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the compound is applied in the dark but becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of ¹²⁵I-labeled α-neurotoxin from Naja naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. About half of the toxin binding sites appear to be blocked by the photoaffinity label.     

Properties of the reaction of GDP-mannose with endogenous polyisoprenylphosphates of liver membranes.

The similarity of yeast microtubular protein to the tubulins purified from other eukaryotic organisms has been open to question. This work involves the identification of two yeast proteins that resemble α and β tubulin prepared from other eukaryots in their ability to copolymerize with purified rat brain tubulin and co-migrate with α and β brain tubulin on a sodium dodecyl sulfate polyacrylamide slab gel.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release

Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15°C or higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 μM for Ca²⁺ and 200 μM for Mg²⁺. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca²⁺, loss of spectrin does not occur. These results suggest that an intact spectrin network is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Histone-like protein and chromatin structure in the wall-less dinoflagellate Gymnodinium nelsoni

Basic nuclear proteins from the wall-less dinoflagellate Gymnodinium nelsoni were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). One major histone-like protein with a molecular weight of about 10 000 was present in acid extracts of whole nuclei and chromatin isolated from growing cultures. In addition, two minor components of 17 000 and 13 000 daltons were also noted. Chromatin fibers spread by the microcentrifugation technique showed no indication of a subunit structure, but instead appeared as smooth threads with a diameter of about 6.5 nm.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Isolation of basolateral and brush-border membranes from the rabbit kidney cortex: Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na⁺ + K⁺)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na⁺ + K⁺)-ATPase activity and [³H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na⁺ + K⁺)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na⁺ and K⁺. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll a-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by <9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is concluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction

Methods are presented for the complete removal of dodecyl sulfate from proteins. Themethods utilize the extraction of dodecyl sulfate anions as ion pairs with triethylammonium or tributylammonium cations into an organic solvent. The protein is insoluble in the organic solvent and is recovered as a precipitate. The methods are applicable to microgram as well as milligram amounts of protein. In all cases studied, the recovery of protein ranges from 70 to 100%. The recovered protein is suitable for N-terminal Edman degradation, tryptic peptide mapping, and amino acid analysis and can be renatured to regain enzymatic activity and antigenicity.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.