Thermodynamics and kinetics of co-operative protein-nucleic acid binding: II. Studies on the binding between protamine and calf thymus DNA

Unspecific binding of a protamine, namely fluorescein-labelled clupeine Z, to double-stranded calf thymus DNA was studied using fluorescence titration methods and chemical relaxation techniques. Both equilibrium and kinetic data have been analysed using general theoretical approaches discussed in the accompanying paper. The results agree well with the predictions made on the basis of a standard co-operative binding model.Basic parameters evaluated are the co-operative binding constant (K), the coefficient measuring co-operative interaction between nearest neighbours (q), the number of nucleotides occupied by one protamine molecule (n) and the rate constant of dissociation at the ends of bound ligand sequences (K_D). Values obtained at 20 °C, pH 7.5 and 0.4 m-NaCl were K = 5.8 × 10⁷m^?1, q = 1700, n = 20 and K_D = 0.29 s^?1. They have been found to be sensitive to the concentration of added salt (NaCl). This effect apparently reflects the essentially electrostatic nature of the binding process. The results can be satisfactorily described in terms of competitive binding of sodium ions.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

Ring cleavage of 2-iminothiazolidine-4-carboxylates by catalytic reduction. A potential method for unblocking peptides formed by specific chemical cleavage at half-cystine residues

Specific chemical cleavage of proteins at S-cyanocysteine residues leads to formation of iminothiazolidinecarboxylyl peptides which, because their amino termini are blocked, are not susceptible to Edman degradation. A catalyst prepared from NiCl₂ and NaBH₄ converts 2-iminothiazolidine-4-carboxylate to alanine and iminothiazolidine carboxylylglycine to Ala-Gly in good yield. Unmodified proteins treated with this catalyst in 8M guanidinium chloride are recovered in good yield, with quantitative conversion of methionine to amino-butyrate and half-cystine to alanine by desulfuration. The catalyst also induces cleavage of a small subclass of peptide bonds, probably Phe-Thr and Phe-Ser sequences, producing discrete fragments.     

Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.     

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that >80% of those cells' DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,000–200,000 daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3′-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells' soluble cytoplasm. The second polymerase, comprising 1.5–3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfhydryl-blocking reagents and has an apparent molecular weight of 49,000. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.     

Regulation of sterol biosynthesis and lysis of cultured hepatoma cells: inhibition of lanosterol demethylation by hydroxysterols

Demethylation of lanosterol by cultured HTC cells is impaired in the presence of 7_α- and 7_β-hydroxycholesterol, 22(R)-hydroxydesmosterol, and miconazole. The toxicity of these hydroxysterols does not correlate with their ability to inhibit lanosterol demethylation or to depress HMG-CoA reductase activity, although parallel changes in the latter two activities suggest that both are modulated by interaction of hydroxysterols with a single cellular target.     

Nuclease-sensitivity of methylated DNA as a probe for chromatin reconstitution by genotoxicants

DNA in Chinese hamster ovary cells was labeled with [¹⁴C]thymidine and [methyl- ³H]-1-methionine in culture, and their nuclei were digested with micrococcal nuclease. Not until 10 percent of bulk DNA was digested did methylated DNA appear in the acid-soluble fraction. When these cells were exposed to UV-radiation, alkylating agents and intercalating agents in culture, the resistance of methylated DNA to digestion by the nuclease was largely or completely eliminated. The change in the sensitivity of methylated DNA to the nuclease indicates a conformational change in chromatin induced by the genotoxicants.     

Purification and properties of two ribonuclease H enzymes from yeast

Two ribonuclease H activities have been purified from Saccharomyces cerevisiae. The major protein, RNase H_A is an acidic protein with a molecular weight of 65,000. RNase H_B is a basic protein with molecular weight of 54,000. Both RNases are active at alkaline pH range and require divalent cations for activity. RNase H_A has an absolute requirement for Mg²⁺, while Mn²⁺ can replace Mg²⁺ for RNase H_B. RNase H_A is inhibited by low concentrations of N-ethylmaleimide, whereas RNase H_B activity is unaffected under similar conditions. Substrate specificity studies using various polyribonucleotide · poly-deoxynucleotide hybrids showed that RNase H_A preferentially degrades polycytidylate, while RNase H_B is specific for polyadenylate. Kinetic analysis of the degradation of specifically end-labeled polymers and analysis of the products of the two yeast RNase H enzymes showed that yeast RNase H_A is an endonuclease producing 5′-phosphorylated oligonucleotides while yeast RNase H_B is a 5′-exonuclease producing 5′-AMP.     

Temperature effect on affinity chromatography of two lectins from the seeds of Ricinus communis

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III_L and III_H, from the seed of Ricinus communis (castor bean) was measured from 7 to 24°C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18°C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ΔG, ΔH, andΔS. It is suggested that the difference in the temperature dependence of the binding energy of these two lectins may be used for their separation at selected temperature.     

Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands

Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Replication of mycoplasmavirus MVL51: VI. Acriflavine stimulates growth of this single-stranded DNA virus.

Acriflavine inhibits the growth of a double stranded DNA mycoplasmavirus, but stimulates the growth of a single stranded DNA mycoplasmavirus. Maximal stimulation occurs when acriflavine is added late during infection and reflects an increased synthesis of viral relative to cellular DNA.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

Purification of spermidine synthase from rat ventral prostate by affinity chromatography on immobilized S-adenosyl(5′)-3-thiopropylamine

A novel affinity chromatographic adsorbent was developed for purification of spermidine synthase from rat prostate. The adsorbent (S-adenosyl(5′)-3-thiopropylamine-Sepharose) possesses a ligand structurally similar to S-adenosyl(5′)-3-methylthiopropylamine (decarboxy AdoMet), a substrate of spermidine synthase. The S-adenosyl(5′)-3-thiopropylamine-Sepharose was prepared by an alkylation on sulfur of S-adenosyl-3-thiopropylamine by bromoacetamidohexyl-Sepharose under mild acidic conditions. The enzyme has been purified to homogeneity in 40% yield by using DEAE-cellulose, affinity chromatography employing S-adenosyl(5′)-3-thiopropylamine-Sepharose, and gel filtration. The enzyme had a molecular weight of approximately 73,000 and was composed of two subunits of equal size. The specificity of the reaction was rather strict, but cadaverine could replace putrescine as the aminopropyl acceptor, and the rate was 1/20th of the rate for spermidine formation. Apparent K_m values for putrescine and decarboxy AdoMet were 0.1 mm and 1.1 μm, respectively. Inhibition by decarboxy AdoMet and 5′-deoxy-5′-methylthioadenosine was observed. The inhibition by 5′-deoxy-5′-methylthioadenosine was partially noncompetitive with respect to decarboxy AdoMet.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.