Incorporation of labelled amino acids by nuclei isolated from Euglena gracilis

Nuclei isolated from the lower eukaryote, Euglena gracilis, incorporate labelled amino acids into hot-TCA-insoluble material under conditions of incubation similar to those used for higher eukaryotes. Optimal temperature for amino acid incorporation by Euglena nucleic is 25° to 30°C. Optimal pH is 5.0. ATP stimulates incorporation only to a small extent. Evidence is presented that the product synthesized is protein.     

Uptake of amino acids and nucleic acid precursors by regenerating rat liver

1. At 0.5-1.0h after partial hepatectomy the intracellular acid-soluble fraction of rat liver took up twice as much radioactivity from [(3)H]orotic acid, [(3)H]uridine and [(3)H]thymidine as did similar fractions from sham-operated animals. This increase in penetration was not prevented by adrenalectomy or actinomycin, both of which decreased precursor uptake into nuclear RNA at this time. The increase in entry was still shown by thymidine at 22h after operation, at the height of the S period. Adenine penetration was not increased 1h after partial hepatectomy. 2. Plasma concentrations of ornithine and possibly methionine, tyrosine and lysine were raised 1.5h after partial hepatectomy. [(3)H]Lysine entry into regenerating liver at this time was increased by 60%; [(3)H]valine uptake was unaffected. Intracellular amounts of tyrosine, phenylalanine and ornithine in the liver were also increased. 3. The relation of these events to the start of liver regeneration is discussed.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Specific measurement of DNA in nuclei and nucleic acids using diaminobenzoic acid

The diaminobenzoic acid (DABA) reaction with DNA, first described by Kissane and Robbins (J. M. Kissane and E. Robbins, 1958, J. Biol. Chem.233, 184–188) and variously modified, was reinvestigated and applied to the measurement of submicrogram quantities of DNA in nuclear fractions and nucleic acid preparations. The reaction conditions were optimized using a small volume of DABA. This method measures 0.1 μg of DNA with a fluorescence twice that of background and is linear to 10 μg of DNA. DABA yeilds a 1000-fold higher fluorescence with DNA compared with RNA, protein, and polysaccharides, and 0.1 μg of DNA is detectable in the presence of 200 μg of RNA or protein. The method is useful for detecting contaminating DNA in RNA preparations prior to hybridization. A simple procedure using ethanol precipitation was developed for removal of common interfering reagents such as sucrose, glycerol, salts, and Triton X-100. Nuclei isolated using detergents and assayed by this method are also free of measurable interfering lipids.     

qNABpredict: Quick,accurate, and taxonomy-aware sequence-based prediction of content of nucleic acid binding amino acids

Protein sequence-based predictors of nucleic acid (NA)-binding include methods that predict NA-binding proteins and NA-binding residues. The residue-level tools produce more details but suffer high computational cost since they must predict every amino acid in the input sequence and rely on multiple sequence alignments. We propose an alternative approach that predicts content (fraction) of the NA-binding residues, offering more information than the protein-level prediction and much shorter runtime than the residue-level tools. Our first-of-its-kind content predictor, qNABpredict, relies on a small, rationally designed and fast-to-compute feature set that represents relevant characteristics extracted from the input sequence and a well-parametrized support vector regression model. We provide two versions of qNABpredict, a taxonomy-agnostic model that can be used for proteins of unknown taxonomic origin and more accurate taxonomy-aware models that are tailored to specific taxonomic kingdoms: archaea, bacteria, eukaryota, and viruses. Empirical tests on a low-similarity test dataset show that qNABpredict is 100 times faster and generates statistically more accurate content predictions when compared to the content extracted from results produced by the residue-level predictors. We also show that qNABpredict's content predictions can be used to improve results generated by the residue-level predictors. We release qNABpredict as a convenient webserver and source code at http://biomine.cs.vcu.edu/servers/qNABpredict/ . This new tool should be particularly useful to predict details of protein–NA interactions for large protein families and proteomes.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Involvement of basic amino acids in the activity of a nucleic acid helix-destabilizing protein

Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein (residues 21-124) increases poly[d(A-T)] Tm and S-peptide (1-20) has no effect on Tm. These results suggest that specific location of several basic amino acids situated on the surface of RNAase A is largely responsible for this protein's DNA melting activity.     

Dynamic properties of interaction between nucleic acid bases and models of amino acids

We have discussed the possible hydrogen bonding mode of adenine-uracil base dimers and peptide side chains, together with their characteristic effects on the stability of adenine-uracil base pairing in chloroform solution by using proton nuclear magnetic resonance methods, where acetamide and butyric acid were used as a model of residues of glutamine and glutamic acid, respectively, and were added to an adenine-uracil equimolar mixture. The stability of base pairing was affected significantly when the model compounds approached the adenine-uracil base pairs; that is, the Hoogsteen type was destroyed, while the Watson-Crick type was formed more favorably.