Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure

A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure.     

The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA

Bacteriophage BF23st(0) DNA was denatured with alkali and fractionated by agarose gel electrophoresis. Seven single-stranded fragments (designated Fragments I--VII) were identified as the major constituents of the phage DNA. The presence of several minor fragments which represent minor populations of the phage genome was also observed. The largest fragment (Fragment I) represents the intact strand of phage DNA, whereas the other fragments form the complementary strand. Thus, BF23st(0) DNA carries single-strand interruptions in only one strand. The arrangement of the major fragments in the nicked strand was determined by use of gamma-exonuclease and agarose gel electrophoresis. From the mode of action of this nuclease, and from the kinetics of release or disappearance of the fragments, the polarity of the fragments in BF23st(0) DNA was specified. In addition, the presence of two types of major phage populations differing in their composition of the fragments was demonstrated. One type has an additional nick (yielding Fragment IV and Fragment V) in a specific fragment (Fragment II) of other type.     

Synthesis of a photoaffinity probe for the beta-adrenergic receptor.

The synthesis and characterization of a beta-adrenergic photo-affinity label, N-(-2-hydroxy-3-naphthoxypropyl)-N′ (-2-nitro-5-azidophenyl ethylenediamine, (NAP-propranolol) is described. The inhibition constants (Ki) for the NAP-propranolol inhibition of ³H-dihydroalprenolol binding and the inhibition of (?)-isoproterenol-stimulated adenylate cyclase in turkey erythrocytes are 100 nM and 19 nM respectively.     

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.     

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.     

Microheterogeneity of the glycoprotein subunit of the (sodium + potassium)-activated adenosine triphosphatase from the electroplax of Electrophorus electricus.

Isoelectric focusing of purified Na,K-ATPase on polyacrylamide gels resolved the protein into ten bands. The catalytic and glycoprotein subunits were separated by sodium dodecyl sulfate gel filtration. Isoelectric focusing of the isolated glycoprotein subunit showed that it accounted for nine of the ten bands. Part of this microheterogeneity can be attributed to variations in sialic acid content in individual bands, since removal of all of the sialic acid by neuraminidase treatment reduced the number of bands to four. It is suggested that the microheterogeneity of the glycoprotein subunit is due to post-translational modifications of oligosaccharides on a common polypeptide backbone.     

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.     

Regulation of glucose-6-p dehydrogenase synthesis in rat epididymal fat pads.

The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

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.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Properties of iron-sulfur protein isolated from Pseudomonas ovalis.

Mutants of $\text{Escherichia coli}$ C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including øX174. Increasingly deficient LPS's from wt and mutant $\text{E. coli}$ C were tested for inactivation of øX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the $\text{E. coli}$ C wt core was found to lead to adsorption resistance towards øX174:

     

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. III. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components.

It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction.

A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

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.