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PubMed1990

Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modified atmospheres and in air.

The development of Listeria monocytogenes Scott A, serotype 4 and aerobic plate counts on minced raw chicken were determined independently at 4, 10 and 27 degrees C. Samples were packaged in flexible film under two modified atmospheres (one containing oxygen and one containing no oxygen) or air. The anaerobic modified atmosphere (75:25, CO2:N2) resulted in the failure of both the aerobic plate counts and L. monocytogenes to grow at all temperatures. Both the L. monocytogenes and aerobic plate counts grew in air at all temperatures. The aerobic modified atmosphere (72.5:22.5:5, CO2:N2:O2), which more closely duplicates commercial practice, inhibited the increase in aerobic plate counts by more than 4 log10 cfu/g compared to air at 4 degrees C. However, the L. monocytogenes was not affected by this atmosphere and increased in numbers by nearly 6 log10 cfu/g at 4 degrees C in 21 days. Regression analysis of the log10 growth and 95% confidence intervals showed that the differences between aerobic plate counts and L. monocytogenes in modified atmosphere were large. The ability of L. monocytogenes to grow in the aerobic modified atmosphere was not affected by level of the L. monocytogenes inoculum nor by the initial level of aerobic plate counts. These data show that modified atmosphere packaging of raw chicken (and probably other meats) can substantially inhibit the aerobic spoilage flora while allowing pathogenic L. monocytogenes to increase.

Animals↗
PubMed1981

The effects of modifiers on enzyme catalysis: a non-classical nearest neighbor approach.

We present a nearest neighbor lattice model of the effects of modifiers on two-state enzyme catalysis of the reaction S in equilibrium with p. We do not in general make the assumptions of the classical approach to cooperative catalysis that yield (1) adsorption isotherms of the same form as those for the corresponding equilibrium system and (2) a rate of the catalyzed reaction proportional to the number of occupied catalytic sites. Closed form results are obtained for two approximations, the Bragg-Williams and the quasi-chemical. The latter requires (1), but is exact for several simple cases, including the concerted model, under this condition. Under (1) it is found that an interaction between modifier and catalytic sites, whether attractive or repulsive, increases the magnitudes of the slopes of the adsorption isotherms but that interactions between identical sites (catalytic or modifier) increase these magnitudes if attractive and decrease them if repulsive. Thus, the former interaction allows for phase transitions if sufficiently attractive or repulsive, but the latter only if sufficiently attractive. Herein also lies the explanation for why the concerted model displays only "positive cooperativity". It is further seen that it is not possible to classify a modifier as an activator or inhibitor of the catalyzed reaction solely on the basis of the sign of the interaction energy between catalytic and modifier sites. For a given energy, the rate of the reaction may increase or decrease in response to the modifier, or it may respond biphasically. Similarly, the rate may respond biphasically to the activities of s or p, leading to instabilities. Thus, possibilities of multiple nonequilibrium stationary states or spatio-temporal patterns are raised.

Enzymes↗
PubMed1992

HPLC determination of a chemically modified nonantimicrobial tetracycline: biological implications.

Chemically modified tetracycline (4-de-dimethylamino tetracycline), like commercially available tetracyclines, is known to inhibit experimentally induced pathologic collagen breakdown. A method for measurement of chemically modified tetracycline in small volumes (50 microliters) of rat serum was developed using reversed-phase HPLC; this was necessary because this tetracycline analog lacks antimicrobial activity and, therefore, cannot be measured with standard bioassays. This method uses the same solution for extraction and elution thus providing a simple and rapid assay for both drugs. Using this technique, the concentration of chemically modified tetracycline and tetracycline were determined in rat serum at different times after oral administration. The serum concentration of chemically modified tetracycline was much higher than that for tetracycline, and its serum half-life was greater. The IC50 of chemically modified tetracycline and tetracycline, as inhibitors of collagenase from rat polymorphonuclear leukocytes, was determined and found to be 4.1 x 10(-8) M (0.02 micrograms/ml) and 2.4 x 10(-4) M (120 micrograms/ml), respectively. Based on the serum levels of these drugs after oral administration, and their IC50 values, chemically modified tetracycline is potentially a far more potent inhibitor of excess collagenase activity than tetracycline, during pathologic conditions, and may have the added advantage of not producing some of the typical complications of long-term antibiotic therapy.

Animals↗
PubMed1985

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues.

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.

Amylose↗
PubMed1986

In vitro biologic responses to native and surface-modified asbestos.

A comparative study was made of in vitro biologic responses to native chrysotile, amosite, and crocidolite and corresponding asbestos fibers whose surfaces were modified by metal oxides. Interferon induction by influenza virus was depressed by approximately 50% by all native asbestos whereas corresponding surface modified asbestos minimally affected this nonspecific cellular defense mechanism. The release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), and lysosomal enzymes, beta-N-acetylglucosaminidase (beta-NAG) and beta-glucuronidase (beta-Gluc), by rat alveolar macrophages after exposure to either native or surface-modified asbestos (which is indicative of membrane damage) was monitored. Although both native and surface-modified asbestos induced significant leakage of LDH, generally, lesser amounts of the enzyme were released as a result of exposure to the latter than to native asbestos. Whereas all forms of native asbestos caused significant release of beta-NAG and beta-Gluc, leakage of these enzymes from macrophages exposed to surface-modified asbestos was minimal. In contrast to native asbestos which induced irritation of cell membranes, as indicated by hemolysis of sheep erythrocytes, surface-modified asbestos exhibited minimal hemolytic activity. The findings indicate that surface modification of different asbestos by metal oxides generally lessened the adverse effect of the native mineral on the aforementioned biologic entities.

Animals↗
PubMed1985

Increased agonist affinity is induced in tetranitromethane-modified muscarinic receptors.

Tetranitromethane (TNM) modifies the muscarinic receptors from rat cerebral cortex. The modified receptor possesses an increased binding affinity (6-9-fold) toward several agonists such as acetylcholine, carbamoylcholine, arecoline, etc. The binding of antagonists (Bmax and Kd) is only slightly altered. The effects of TNM treatment can be prevented by atropine, thus indicating that TNM modifies residue(s) at the binding site. We carried out a series of successive chemical modifications which indicated that the modified residue(s) is (are) most probably a tyrosyl and not a cysteinyl residue. This conclusion gains support from the pH profile of agonist binding, which suggests the involvement of a residue with an apparent pK comparable to that of the phenolic hydroxyl of a nitrotyrosyl residue. The binding properties of the modified receptor, when compared to those of the native one, clearly indicate that the response to TNM modification with respect to the binding of agonists such as acetylcholine and carbamoylcholine is different from that when oxotremorine and its analogue are employed. This is interpreted as being the result of different binding modes exhibited by the various agonists. Nitration of the receptors can be prevented by the presence of an antagonist but not by an agonist. We propose that this differential response is due to the formation of ligand-receptor complexes that differ with respect to the microenvironment of the modified tyrosyl residue.

Acetylcholine↗
PubMed1986

Properties of chemically modified protein S: effect of the conversion of gamma-carboxyglutamic acid to gamma-methyleneglutamic acid on functional properties.

Protein S, the protein cofactor for activated protein C in the proteolytic inactivation of factor Va, was chemically modified with a mixture of morpholine and formaldehyde. This treatment resulted in the conversion of the gamma-carboxyglutamic acid (Gla) residues of this vitamin K dependent protein to gamma-methyleneglutamic acid. With a 10,000-fold molar excess of morpholine and formaldehyde over protein S it was found that between 10 and 11 Gla residues could be modified. The degree of modification was proportional to the concentration of the modifying reagents used. The modification of as few as two residues resulted in the 70% loss of activity. Calcium inhibited the modification of several residues. In the presence of 3.2 mM calcium ion, a derivative with 2.5 residues modified was prepared that appeared to have full activity. Modification of protein S resulted in the alteration of a number of its properties. The quenching of intrinsic fluorescence by calcium decreased. The quenching effect of terbium ions was also decreased. However, the modified protein and the native protein were equivalent when protein-dependent terbium fluorescence was measured. When modified, protein S would no longer bind to phospholipid vesicles. Finally, the ability of protein S to self-associate was decreased by modification. These findings suggest that the gamma-carboxyglutamic acid residues of protein S may play several roles in the maintenance of structure.

1-Carboxyglutamic Acid↗
PubMed1977

Thermodynamics of binding to native alpha-chymotrypsin and to forms of alpha-chymotrypsin in which catalytically essential residues are modified; a study of "productive" and "nonproductive" associations.

The standard free energy (deltaG degrees), enthalpy (deltaH degrees), and entropy (deltaS degrees) of association for proflavin and D- and L-N-AcTrp have been obtained at pH 7.8 for native alpha-chymotrypsin (Cht) and for forms of Cht in which essential catalytic residues of the active site are modified. The modified Cht forms studied are dehydroalaninyl-195-alpha-Cht and N-methylhistidinyl-57-alpha-Cht. Associations to native Cht (pH 7.8) are characterized by negative deltaH degrees and deltaS degrees values (i.e., for L-AcTrp deltaH degrees = -9.1 kcal/mol and deltaS degrees = -21 eu at T = 25 degreesC). In contrast, we found associations to modified Chts to be characterized by an enthalpy near zero and a positive entropy of association, the values of the deltaH degrees and deltaS degrees for association to the modified Cht forms being similar to those expected for transfer of small aromatic molecules from water to a nonpolar solvent phase. Differences in deltaH degrees and deltaS degrees observed for binding of substrate analogues and inhibitors to modified and native Cht (pH 7.8) are approximately + 10 kcal/mol and +30 eu, respectively. Data from D. D. F. Shiao ((1970), Biochemistry 9, 1083) similarly show differences of comparable magnitude between binding of substrate analogues to active alpha-Cht (pH 7.8) and the His-57 protonated form of alpha-Cht (pH 5.6). The negative deltaH degrees and deltaS degrees values of associations for binding to active alpha-Cht indicate that a substrate-induced conformational change occurs on substrate association with the primary binding site (S1), which does not occur in Ser-195 and His-57 modified Cht. From these differences we infer a linkage between binding of substrate into S1 and the catalytic residues in the nucleophilic subsite (S1-S1'). Our data also show that associations of substrate analogues into potentially productive Michaelis complexes S1 cannot be easily differentiated from associations that are nonproductive (i.e., nonactivated) from their deltaG degrees obsd, but may be differentiated by their respective deltaH degrees obsd and deltaS degrees obsd for association. Accordingly, it is indicated that the probable substrate association-activation process, characterized thermodynamically in this work, occurs in the substrate binding step and leads to lowered free energies of activation in catalytic steps succeeding binding however, the process does not influence the observed strength of substrate binding.

Binding Sites↗
PubMed1977

Conformation of DNA modified with a dihydrodiol epoxide derivative of benzo[a]pyrene.

The conformation of calf thymus DNA modified by reaction with (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, which binds covalently mainly to the 2-amino group of guanosine residues, was studied. With samples in which 1.5 or 2.2% of the bases were modified, there was a slight decrease in Tm during heat denaturation and a slight increase in susceptibility to the single strand specific nuclease S1. In a DNA sample in which 4.5% of the bases were modified, there was an appreciable decrease in Tm and a marked increase in susceptibility to S1 nuclease. The kinetics of the reaction of the modified DNAs with formaldehyde provided evidence for locally destabilized regions ranging from 1 to 7 base plates, depending on the extent of modification. Alkaline and neutral sucrose gradient analyses revealed no evidence for strand breakage in the 1.5 and 2.2% modified samples, although single-strand breaks were found in the 4.5% modified samples. Taken together, these results suggest that DNA molecules containing a covalently bound benzo[a]pyrene derivative have an altered conformation characterized by small localized regions which are destabilized and easily denatured. The conformational changes associated with the covalent binding of the benzo[a]pyrene derivative to native DNA appear to be different from, and less marked, than those associated with the covalent binding of N-2-acetylaminofluorene to native DNA.

Animals↗
PubMedDate not supplied

Synthesis, characterization, and solution properties of ras sequences modified by arylamine carcinogens at the first base of codon 61.

The complementary pentadecamers d(5'-TACTCTTCTTGACCT) (strand A) and d(5'-AGGTCAAGAAGAGTA) (strand B), which span a portion of the mouse c-Ha-ras protooncogene centered around codon 61, were synthesized by using standard beta-cyanoethyl phosphoramidite chemistry and characterized by sequence analysis. Strand A, containing a sole guanine at the position corresponding to the first base of codon 61, was modified with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene or its 4-aminobiphenyl analogue. In both cases only the corresponding N-(deoxyguanosin-8-yl)arylamine adduct was formed, as judged from HPLC and UV analyses conducted after enzymatic hydrolysis of the modified oligomers. Nonmodified and modified pentadecamers were annealed with strand B. Cooperative melting transitions were observed with all samples, thus indicating the formation of stable duplexes. Melting temperatures decreased in the order nonmodified duplex greater than 2-aminofluorene-modified duplex greater than 4-aminobiphenyl-modified duplex, which indicated destabilization of the helical structure upon incorporation of the adducts, with 4-aminobiphenyl having the greatest effect. Circular dichroism spectra of all duplexes were characteristic of an overall right-handed B-type conformation, with no major conformational differences being detected between the two arylamine-modified oligomers.

Aminobiphenyl Compounds↗
PubMed1990

Comparative study of chemical modifiers for the determination of molybdenum in milk by electrothermal atomisation atomic absorption spectrometry.

A comparative study of various chemical modifiers for the determination of molybdenum in milk by electrothermal atomisation atomic absorption spectrometry was carried out. Methods with nitric acid or barium difluoride as the chemical modifier and in the absence of a chemical modifier were studied by introducing the milk samples directly into the graphite furnace with octyl alcohol. The graphite furnace programme, amount of modifier and the calibration and additions graphs were studied in all instances. The characteristic masses were 17.82, 18.64 and 12.08 pg of molybdenum in the absence of a chemical modifier and with nitric acid or barium difluoride as the chemical modifier, respectively. The precision, accuracy and interferences of the method were also investigated.

Animals↗
PubMed1984

The low-density-lipoprotein pathway of native and chemically modified low-density lipoproteins isolated from plasma incubated in vitro.

Normal fasting human plasma was incubated for 24 h at 37 degrees C in the presence or absence of lecithin:cholesterol acyltransferase (LCAT) inhibitors. The low-density lipoprotein (LDL) fractions of incubated plasma (control LDL and LCAT-modified LDL) were studied with respect to their chemical and functional properties. LCAT-modified LDL differed from control LDL by a decreased phospholipid and free-cholesterol content, but increased cholesteryl esters. Furthermore, an increase of the relative protein content in LDL by 16-20% was found. Apolipoproteins of LCAT-modified LDL exhibited a 10-fold increase of apo AI, a 4-5-fold increase of apo E, and a 2-fold increase of apo C. All these apolipoproteins resided together with apo B on the same particles. LCAT-modified LDL displayed a higher electrophoretic mobility, a higher hydrated density, a decreased flotation constant and a smaller diameter. Cultured human fibroblasts bound and internalized LCAT-modified LDL to a lower extent than control LDL. The degradation, however, was faster. Modified LDL suppressed 3-hydroxy-3-methylglutaryl-CoA reductase activity to a lower extent than did control LDL. Our results demonstrate that LCAT action, together with lipid transfer and exchange processes, markedly alters the chemical and physiochemical properties of LDL. This in turn significantly influences LDL catabolism in vitro.

Cells, Cultured↗
PubMed1987

Some electron-transfer reactions involving carbodi-imide-modified cytochrome c.

The reaction kinetics of native and carbodi-imide-modified tuna and horse heart cytochromes c with both a strong (dithionite) and a relatively weak (ascorbate) reducing agent were studied over a wide range of conditions. In their reactions with dithionite both the native and modified cytochromes exhibit single exponential time courses. The effects of dithionite concentration and ionic strength on the rate of the reduction are complex and can best be explained in terms of the model proposed by Lambeth & Palmer [(1973) J. Biol. Chem. 248, 6095-6103]. According to this model, at low ionic strength the native proteins are reduced almost exclusively by S2O4(2-) whereas the modified proteins showed reactivity towards both S2O4(2-) and SO2.-. These findings are interpreted in terms of the different charge characteristics of the carbodi-imide-modified proteins relative to the native proteins. The findings that the modified proteins react with ascorbate in a biphasic manner are explained as arising from ascorbate binding to a reducible form of the protein, before electron transfer, with an equilibrium between the ascorbate-reducible form of the protein and a non-reducible form. Estimates were obtained for both the ascorbate equilibrium binding constant and the rate constant for the internal electron transfer for both the native and modified horse and tuna proteins. The effect of pH on the reactions indicates that the active reductant in all cases is ascorbate2-. The studies of ascorbate reactivity yield important information concerning the proposed correlation between ascorbate reducibility and the presence of a 695 nm-absorption band, and the study of dithionite reactivity illustrates the effect of protein charge and solution ionic strength on the relative contributions made by the species SO2.- and S2O4(2-) to the reduction of ferricytochrome c.

Animals↗
PubMed1990

Kinetic analysis of regeneration by dilution of a covalently modified protein.

An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.

Adsorption↗
PubMed1990

Utilization of the free energy of the reversible binding of protein and modifying agent towards the rate-enhancement of protein covalent modification.

An analysis is presented of the catalytic factors responsible for the rate-enhancement that may be observed when a protein modification reaction is compared with a reaction of the same modifying agent with a model micromolecular compound exhibiting the same reactive group as the protein under study. It is seen that affinity-mediated rate-enhancement of protein modification is realized by the loss of activation entropy. On the assumption that attainment of maximal affinity-mediated rate-enhancement presents with an activation entropy of the protein modification reaction equal to zero, whereas the activation enthalpy of the reaction remains unchanged, it is shown that the value for maximal affinity-mediated rate-enhancement is equal to e-delta s++/R. Accordingly, protein modification reactions may be differentiated into (i) reactions the rate-enhancement of which (relative to the reaction of the same modifying agent with a model compound) is primarily entropy-controlled and (ii) reactions the rate-enhancement of which is primarily enthalpy-controlled. It is seen that modifying agents of low reactivity towards model compounds, but with a high, i.e. highly negative, activation entropy are better suited as prospective affinity-based protein-modifying agents, since the potential affinity-mediated rate-enhancement, and hence the selectivity, of these compounds is necessarily high. Kinetic and thermodynamic constants of the reaction of modifying agents with proteins, and with model compounds, and values of maximal affinity-mediated rate-enhancement, based on published data of the reaction of several modifying agents with model compounds, are presented and discussed.

Kinetics↗
PubMed1985

Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein.

Cultured rabbit and bovine aortic endothelial cells generated chemotactic activity for mouse resident peritoneal macrophages, demonstrable in the conditioned medium. This chemotactic activity was heat stable and was not extracted into chloroform/methanol. It was inhibited by addition of endothelial cell-modified low density lipoprotein (EC-modified LDL), a form of LDL shown previously to contain peroxidized lipids, increased lysophosphatidylcholine, and partially degraded apoprotein B. The chemotactic activity was also inhibited by LDL previously oxidized in the absence of cells with 5 microM Cu2+. Inhibitory activity was present in the lipid extract of EC-modified LDL but not in that of native LDL, presumably representing peroxidized lipid components. EC-modified LDL also inhibited the chemotactic activity of zymosan-activated serum. Because EC-modified LDL is taken up in part by way of the acetyl-LDL receptor, the effects of acetyl-LDL were tested. Rather than inhibiting chemotaxis, acetyl LDL showed intrinsic positive chemotactic activity as did also fucoidin and polyinosinic acid, both of which also interact with the acetyl-LDL receptor. These studies suggest mechanisms by which macrophages may be recruited into the subendothelial space by endothelial cell-derived chemotactic factors or by natural polyanions structurally related to fucoidin or polyinosinic acid and then become "trapped" there because of the inhibitory effects of peroxidized lipid components in modified forms of LDL.

Animals↗
PubMed1987

Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II).

Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemotherapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)b, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited (50% control) in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations (600-900 fmol) of cis-DDP bound to DNA, (D/N)b = 0.031. Similar concentrations (4-6 nM) of cis-DDP-modified poly(dG).poly(dC) also inhibited antibody binding, whereas higher concentrations (17-36 nM) of cis-DDP-modified poly[d(AG)].poly[d(TC)] were required for inhibition. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl2], [Pt(dach)Cl2], and [Pt(NH3)2(cbdca)] (carboplatin) (where en is ethylenediamine, dach is 1,2-diaminocyclohexane, and cbdca is cyclobutane-1,1-dicarboxylate) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA, (D/N)b = 0.022. This antibody crossreacted with unmodified, denatured DNA. Its binding to trans-DDP-DNA was selectively inhibited by trans-DDP-modified poly[d(GT)].poly[d(CA)] (50% inhibition at 1 nM bound trans-DDP). The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities.

Animals↗
PubMed1989

Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.

Bacteriophage lambda↗