Biomedical subjects

K S Matthews

Publications and source records attributed to K S Matthews.

At least 91 records · Page 5Linked to original sources

PubMed1986

Thermodynamic analysis of the lactose repressor-operator DNA interaction.

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.

Escherichia coli↗

PubMed1986

Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation.

Aphidicolin↗

PubMed1986

Correlation of immunologic and nutritional status with infectious complications after major abdominal trauma.

Examination of the response of injured patients' lymphocytes to the mitogen phytohemagglutin in a defined medium provides a mechanism to define the relationship between alteration in immune function and septic complications. Lymphocytes from 30 victims of gunshot wounds to the abdomen were examined. Response to mitogen was measured by incorporation of [3H]-thymidine as a function of lymphocyte concentration, with a constant amount of mitogen phytohemagglutinin and a standard incubation period. A saturation curve was obtained, and lymphocyte response was expressed as the concentration necessary for half-maximal incorporation of radioactive label, L1/2. Lymphocyte transformation was compared with that found in a group of 50 healthy volunteers. On arrival in the emergency center, the in vitro lymphocyte response of patients was markedly diminished. There were seven patients for whom a lymphocyte curve could not be generated, i.e., L1/2 greater than 1 X 10(6). For the other 23 patients, L1/2 = 4.75 X 10(5) (SEM - 7.5 X 10(4)) compared with L1/2 = 1.5 X 10(5) (SEM - 5 X 10(4)) for normal volunteers (p less than 0.01). Measurement of skin test response, white blood cell count, anthropometric measurements, and albumin level were not predictive of patient course. In contrast the in vitro lymphocyte viability corresponded to the degree of injury, and recovery of lymphocyte function was associated with improvement in the patient's clinical course.

Abdominal Injuries↗

PubMed1985

Thermal denaturation of the core protein of lac repressor.

The thermal denaturation of the core protein of lac repressor was studied alone and in the presence of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the antiinducer o-nitrophenyl beta-D-fucoside (ONPF) by means of high-sensitivity differential scanning calorimetry. The denaturation that takes place at about 65 degrees C is apparently irreversible; i.e., a rescan of a previously scanned sample of protein solution shows no denaturational endotherm. Despite this irreversibility, the denaturation appeared to follow quantitatively the dictates of equilibrium thermodynamics as embodied in the van't Hoff equation. The results obtained indicate clearly that the tetrameric protein dissociates to monomers during denaturation and that the ligands are not dissociated until denaturation takes place. The enthalpy of denaturation of the protein is 4.57 +/- 0.25 cal g-1 and is independent of temperature. The enthalpies of dissociation of IPTG and ONPF at the denaturation temperature are very large, 37 and 42 kcal (mol of ligand)-1, respectively.

Calorimetry, Differential Scanning↗

PubMed1985

Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties.

Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.

Carbon Radioisotopes↗

PubMed1985

A mutant lactose repressor with altered inducer and operator binding parameters.

The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine. Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex. Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation. The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins. This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts. Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.

Acetamides↗

PubMed1985

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein.

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.

Amino Acid Sequence↗

PubMed1984

Trinitrobenzenesulfonate modification of the lysine residues in lactose repressor protein.

Modification of the lysine residues in the lactose repressor protein has been carried out with trinitrobenzenesulfonate. Reaction of lysine residues at positions 33, 37, 108, 290, and 327 was observed. Inducer binding was increased by modification with this reagent, while both nonspecific DNA binding and operator DNA binding were diminished, although to differing degrees. The loss in operator DNA binding capacity was complete with modification of approximately 2 equiv of lysine per monomer. The extent of reaction was affected by the presence of both sugar and DNA ligands; binding activities of the modified protein and reaction pattern of the lysines were perturbed by these ligands. The presence of operator or nonspecific DNA during the reaction protected against specific and nonspecific DNA binding activity loss. This protection presumably occurs by steric restriction of reagent access to lysine residues which are essential for both nonspecific and operator binding interactions. Lysines-33 and -108 were protected from modification in the presence of DNA. These experiments suggest that the charge on the lysine residues is important for protein interaction with DNA and that steric constraints for operator DNA interaction with the protein are more restrictive than for nonspecific DNA binding. In contrast, inducer (isopropyl beta-D-thiogalactoside) presence partially protected lysine-290 from modification while significantly enhancing reaction at lysine-327. Conformational alterations consequent to inducer binding are apparently reflected in these altered lysine reactivities.

Chromatography, High Pressure Liquid↗

PubMed1984

lac operator DNA modification in the presence of proteolytic fragments of the repressor protein.

Singly end-labeled DNA fragments containing the lactose operator were methylated in the presence of the lactose repressor and homogeneous preparations of its proteolytic fragments. Binding of core protein produced by mild trypsin digestion yielded a methylation perturbation pattern that differed significantly from that elicited by binding to intact repressor, although similarities in the patterns for these related proteins were noted in the central, asymmetric region of the operator. An NH2-terminal peptide (residues 1 to 56) from lac repressor bound operator fragments in a nitrocellulose filter assay, but failed to perturb DNA methylation significantly relative to the pattern in the absence of peptide. Binding of hybrid tetramers of core and intact repressor monomers produced related but unique methylation patterns for the purines on the operator fragment. The general pattern of perturbation observed suggests preferred binding of a single NH2 terminus to the promoter-distal region of the operator and asymmetric interaction of the core region with the operator sequence. Differences in purine methylation patterns produced by the presence of effector complexes of repressor and core protein suggest the possible nature of changes in protein topology that result in the affinity changes accompanying induction.

Alkylating Agents↗

PubMed1984

Enzymatic digestion of operator DNA in the presence of the lac repressor tryptic core.

The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3' boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3' boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.

Autoradiography↗

PubMed1984

Chemical modification of dopamine beta-hydroxylase.

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.

Amino Acids↗

PubMed1983

Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.

Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.

DNA, Bacterial↗

PubMed1981

Tetranitromethane modification of the tyrosine residues of the lactose repressor.

Repressor protein modified with N-ethylmaleimide has been used to determine the exclusive effects of tyrosine nitration by tetranitromethane. Since modification of proteins with tetranitromethane generally results in both cysteine oxidation and tyrosine nitration, N-ethylmaleimide has been used to protect the cysteines in the repressor against oxidation in subsequent tetranitromethane reactions. Nitration of tyrosine residues in repressor previously reacted with N-ethylmaleimide results in loss of both specific and nonspecific DNA-binding activities. Na2S2O4 reduction of tetranitromethane-modified protein restores partial operator DNA-binding and complete nonspecific DNA-binding capability. Residues primarily affected are tyrosines 7 and 17, which are both in the NH2 terminus. Inter- and intramolecular cross-links which are observed in the modified protein can be minimized by altering reaction conditions; the cross-links present occur between sites located in the NH2 termini. Modification of the core protein also results in loss of the operator DNA-binding capacity, and subsequent reduction restores partial operator-binding activity. Both operator and nonspecific DNA-binding capabilities of the repressor protein are protected by the presence of nonspecific DNA during the tetranitromethane modification, and simultaneously the extent of nitration is decreased.

Binding Sites↗

PubMed1981

Determination of the ligand-binding characteristics of several tight-binding mutants of the lactose repressor protein.

Several tight-binding mutants of the lactose repressor protein have been characterized with respect to their fluorescence properties and their inducer, operator and nonspecific DNA-binding constants. The tryptophan fluorescence emission spectra for the mutants and the wild-type repressor are quite similar. However, alterations in the Stern-Volmer constants for iodide quenching of the tryptophans in the mutant proteins compared to wild-type suggest differences in the local environment or solvent accessibility for these amino acids in the tight-binding repressors. The inducer-binding affinities and association rate constants of the mutant proteins and protein-operator DNA fragment complexes are also altered compared to wild-type. The extents of these changes vary among the different mutant repressors. The nonspecific DNA-binding affinities of the mutant proteins are 2--3-fold greater than the wild-type repressor, and the affinities of the tight-binding proteins for a 29 base-pair operator DNA fragment are also increased, though to a varying extent depending upon the mutant. The phenotypic behavior of these proteins in vivo can be partially explained by these results obtained in vitro; however, it is likely that there are additional factors responsible for the tight-binding behavior of the proteins that were not detectable in these experiments.

Alleles↗

PubMed1980

DNA binding characteristics of lactose repressor and the trypsin-resistant core repressor.

The nonspecific DNA binding capacity of repressor protein has been assessed by boundary sedimentation of repressor and calf thymus DNA fragmented by shearing and by nitrocellulose ultrafiltration employing labeled lambdaplac DNA in the presence of inducer concentrations sufficient to insure dissociation of repressor from the operator region of the DNA. These methods gave values in good agreement with values previously reported in the literature. The association constants for the interaction of repressor with operator DNA fragments and lambdaplac DNA have been measured and found to differ by approximately 100-fold at low salt concentrations, but the difference decreases to 4-fold at salt concentrations near the physiological value. The equilibrium association constant for the repressor-operator DNA fragment is significantly less sensitive to salt concentration than the corresponding constant for lambdaplac DNA. Inducer decreases the salt concentration dependence of repressor-operator DNA fragment only slightly. Measurement of the association constants for the interaction of the trypsin-resistant core protein with operator DNA fragment and lambdaplac DNA indicate that the core protein binds to the two DNA's with the same affinity. This result contrasts with the differential affinity of intact repressor for these two DNA's. In addition, the core protein association constants for operator DNA fragment and lambdaplac DNA display minimal dependence on the salt concentration. These results suggest a role for nonionic interactions in the binding of core protein to operator DNA.

Animals↗

PubMed1980

Equilibrium binding of inducer to lac repressor.operator DNA complex.

The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases the affinity of the protein for inducer molecules. The maximum change observed is a 20-fold increase in the concentration of inducer necessary for half-saturation of the protein. The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating amounts of operator DNA fragments were compared to curves predicted by several different models. Using the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to discriminate between the various models. The models which accurately predicted the inducer binding curves and conformed to the measured values for the other parameters were Monod-Wyman-Changeux and Koshland type models with the following features: 1) positive cooperativity in the binding of inducer to the repressor . operator DNA complex; 2) two sites for the binding of the operator DNA fragment to repressor protein; 3) effect of binding of each operator DNA fragment on all four subunits of the repressor. These models are consistent with all of the available data, and they indicate that the binding of the first two inducer molecules accounts for greater than or equal to 60% of the difference in affinities between free and induced repressor for operator DNA; in vivo this difference should be sufficient for induction to occur.

DNA, Bacterial↗

PubMed1980

Kinetic studies of inducer binding to lac repressor.operator complex.

The rates of binding of inducer molecules to the lactose repressor protein are significantly affected by the presence of bound operator DNA fragments. The association rate is decreased from 4.6 x 10(4) M-1S-1 to 1.0 x 10(4) M-1S-1 by the presence of saturating amounts of operator DNA fragments. The inducer dissociation rate was measured by dilution of repressor . operator . inducer complexes and by displacement of the sugar molecule from repressor by the binding of operator DNA. The value for the dissociation rate in the presence of bound DNA was 0.8 S-1; this is 4-fold greater than the inducer dissociation rate measured in the absence of operator. These kinetic results suggest that repressor should exhibit a 20-fold lower affinity for inducer when operator DNA fragments are bound; this prediction is in agreement with previous equilibrium measurements. Measurement of the rate of operator fragment dissociation from repressor using a nitrocellulose filter assay yields a value of 0.04 S-1 in the absence of inducer; this dissociation rate is too rapid to measure in the presence of bound inducer molecules. Calculations assuming that the DNA-protein association rate is unaffected by sugar binding suggest that the rate of dissociation of DNA from repressor containing 4 bound inducer molecules may be as high as 40 S-1. Determination of the apparent association rate for inducer binding to operator DNA fragments indicates that DNA molecules do dissociate from the protein . operator . inducer complex at intermediate stages during the sugar binding reaction. The exact point at which dissociation occurs at a complex function of the concentrations of operator and inducer and the relationship between the rates for sugar binding and for operator fragment dissociation which are comparable for some of the protein species.

Allosteric Regulation↗

PubMed1980

Hybrid tetramers of native and core lactose repressor protein. Assessment of operator and nonspecific DNA binding parameters and their relationship.

Hybrid tetramers of lac repressor and its trypsin-resistant core protein were produced by mild proteolytic digestion and isolated by chromatography on phosphocellulose. These tetramers were used in binding studies to probe the relationship of the NH2 terminus and core domains in operator DNA binding and to explore subunit participation in both operator and nonspecific DNA binding. The purity of each tetramer was demonstrated by several lines of evidence, including physical characterization and measurement of the binding activities of the hybrid tetramer preparations. Each tetramer displayed measurable operator binding activity, and the dissociation constants of the tetramers for lambdaplac DNA and a 29 base operator fragment increase with the loss of each NH2 terminus. These studies strongly suggest that each NH2 terminus of the repressor interacts with the operator DNA, although the four NH2 termini do not appear to make equal contacts. The contributions of the NH2 terminus to operator binding do no appear to be identical with those for nonspecific DNA binding. In addition, the binding of the NH2 termini to nonspecific DNA can be described by postulating four identical, noninteracting sites. Although the rate of dissociation of the repressor-operator is increased by a factor of 2 upon removal of each NH2 terminus, calculations using the measured dissociation rate and equilibrium dissociation constant indicate that the rate of association of repressor to operator is decreased approximately 5-fold. These data suggest that the contacts made by the NH2 termini may greatly facilitate association with DNA. Competition studies of nonspecific DNA with operator DNA binding confirm the existence of two operator DNA binding sites on each tetramer and suggest that the contacts of a single NH2 terminus are not identical for the two operator binding sites.

Binding, Competitive↗