Affect Protein Binding Research Articles

Cisapride. Drug interactions of clinical significance.

Cisapride is a prokinetic agent which restores motility of the gastrointestinal tract in conditions of decreased bowel transit. It may also alter the absorption of coadministered drugs. The absorption of morphine, diazepam, cyclosporin, alcohol (ethanol) and levodopa are increased. Initial absorption of cimetidine and raniditine is also increased, but overall absorption is lower due to increased bowel transit. The absorption of digoxin, propranolol and the anticoagulants warfarin and phenprocoumon appears unaffected by cisapride, although increase thrombotest values were seen with acenocoumarol (nicoumalone). Drug interactions leading to increased plasma concentrations of cisapride may produce an increase in adverse effects. The most important of these is QT interval prolongation and ventricular arrhythmias. Phenytoin does not appear to affect protein binding of cisapride. Cisapride metabolism is inhibited by the antifungals ketoconazole, fluconazole, itraconazole and miconazole, and by the antibacterials erythromycin, troleandomycin and clarithromycin. Cisapride should not be coadministered with these drugs. Cimetidine produces a small increase in cisapride plasma concentrations, which may be due to inhibition of metabolism. Cisapride absorption is unaffected by other antacids. Atropine may reverse the cisapride-induced increase in peristalsis. Prescribers should remain vigilant to the presence of these and other, as yet unreported, reactions.

The Development and Use of a DNA Polymerase Arrest Assay for the Evaluation of Parameters Affecting Intrastrand Tetraplex Formation

We show here that a K+-dependent block to DNA synthesis is a sensitive and specific indicator of intrastrand tetraplex formation that can be used, both to identify sequences with tetraplex-forming potential and to examine parameters that affect tetraplex formation. We show that tetraplex formation is determined by a complex combination of factors including the size and base composition of its constituent loops and stems. In the process of carrying out this study we have found that the number of sequences with the ability to form tetraplexes is larger than previously thought, and that such sequences are ubiquitous in eukaryote genomes.

Thermal Gelation of Stretched and Cold‐Shortened Bovine Sternomandibularis Muscle and Myofibrils

ABSTRACTMuscle gels (10% protein) and myofibril gels (8% protein) were prepared at pH 6.0 with 2% NaCl and a heating rate of 0.7°C/min. No difference in gel strength occurred between stretched and cold‐shortened muscles, but cooking loss was lower for stretched muscle. Stretched muscle sarcomeres were longer than those of cold‐shortened muscle. The myofibril fraction from stretched muscle had higher gel strength, viscosity index, elasticity, and lower cooking loss than that from cold‐shortened muscle. These results suggest that the contractile state of the muscle affects protein binding and water binding of the myofibrillar fraction.

Inhibition of nuclear protein binding to two sites in the murine c-myc promoter by intermolecular triplex formation.

The c-myc gene is overexpressed in a variety of tumor types and appears to play an important role in the abnormal growth of a number of cell types. In an effort to determine the ability of sequence- and species-specific triplex-forming oligonucleotides to inhibit expression of a targeted gene in animals, we have identified two novel triplex-forming sites in the murine c-myc promoter. One is homologous to the triplex-forming human PuF binding element located upstream of the P1 transcription start site. The other triplex-forming site is found in a region between P1 and P2 that encompasses the ME1a1 binding site and part of the E2F binding site and is highly homologous to the human sequence. Synthetic oligodeoxyribonucleotides designed to target these essential regulatory elements form sequence-specific triple helices as demonstrated by gel mobility shift analysis and DNase I footprinting. Polypurine: polypyrimidine regions in the P1 and P2 promoters form specific protein-DNA complexes upon incubation with a murine YC8 nuclear extract. Preincubation of each of the promoter fragments with its respective triplex-forming oligonucleotide results in the inhibition of nuclear protein binding. Non-triplex-forming oligonucleotides do not significantly affect protein binding. The data presented are a preliminary step toward generating an animal model for the phenotypic effects of triplex formation within the c-myc promoter.

Complex functional interactions at the early enhancer of the PQ strain of BK virus.

BK virus is a human papovavirus that latently infects a majority of the world's population. There are more than 30 strains of the virus, most of which differ in the structure of the early enhancer region. The enhancer of the progenitor strain, WW, from which the other strains can be derived, consists of four conserved DNA domains, P, Q, R, and S. Rearrangement of the enhancer occurs upon passage in tissue culture and is thought to occur during virus replication. The strain under study, PQ, was selected upon passage of the Gardner strain (PPPQS) in the permissive cell line, Vero. Mutational analysis of the entire enhancer region demonstrates the importance of five cis-acting sequences: DNA sites B, C, and F, which have homology to the NF-1 protein binding sequence; one purine-rich motif designated A; and site D, which is similar to an SP-1 protein binding site. Two sites, B and C, appear to have a negative influence on gene activity. To study the functional interactions in more detail, promoter-enhancer constructions that contain different combinations of the five DNA sites linked to the chloramphenicol acetyltransferase gene were tested for early gene activity. The results reveal that the proteins binding to the enhancer functionally cooperate with each other. The effects of making mutations at the DNA sites are very similar to the effects of using excess enhancer DNA sequences to titrate the proteins that bind to the cis-acting DNA sites (in vivo competition). Moreover, the effects of changing the spacing between the DNA sites also demonstrate that there are cooperative interactions among the proteins that bind to the PQ strain enhancer. DNA sites B, C, and F are clearly protected from DNase I digestion by Vero cell nuclear proteins. In addition, mutation of each DNA site alters its sensitivity to DNase I in the presence of Vero cell proteins. Interestingly, mutation of site B affects protein binding to site B as well as to sites A, C, D, and F. These results suggest that cooperative functional and physical interactions occur at the early enhancer of the PQ strain.

Methylation of an α-foetoprotein gene intragenic site modulates gene activity

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding.

Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity

We report measurements of the relative binding affinity of CAP for DNA sequences which have been systematically mutated in the region flanking the consensus binding site. Our experiments focus on the locus one helical turn from the dyad axis where DNA bending toward the minor groove is induced upon C-AP binding. The binding free energy and extent of bending are moderately well correlated for the set of 56 sequences. Changes in binding affinity spanning a factor of about 50 could be accounted for by additive contributions of dinucleotides; with a few exceptions, the relative ranking of dinucleotide contributions to binding and bending are similar. We conclude that dinucleotides are the smallest independent unit required for quantitative interpretation of CAP-induced DNA bending and binding in the distal domains of the CAP consensus binding site. The imperfect correlation between binding strength and extent of bending implies that sequence changes affect protein binding strength not only by altering the DNA deformation energy required to form the complex, but also by affecting directly the free energy of interaction between protein and DNA.

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1

Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene.

Recent developments in the study of the effects of cigarette smoking on clinical pharmacokinetics and clinical pharmacodynamics.

With the ever-increasing population of cigarette smokers, the potential for cigarette smoke to affect drug therapy both pharmacokinetically and pharmacodynamically is significant. The overriding pharmacokinetic effect is increased drug metabolism through the induction of liver enzymes. The constituents of tobacco smoke, primarily nicotine, have their own pharmacological effects which may potentiate or antagonise the desired pharmacological effect of a particular drug, thereby affecting its efficacy. Furthermore, end-organ responsiveness may also be altered by tobacco. These latter 2 aspects constitute altered clinical pharmacodynamics. Approximately 30 drugs have been evaluated in terms of cigarette smoking. Induction of liver enzymes has been shown to increase the metabolism of imipramine, meprobamate, oestrogens, pentazocine, phenylbutazone, theophylline and warfarin. Nicotine has been shown to inhibit diuresis, alter ulcer healing, impair subcutaneous absorption, affect protein binding and stimulate catecholamine release; these effects have been evaluated in terms of therapy with frusemide (furosemide), histamine H2-antagonists, insulin, lignocaine (lidocaine) and beta-blockers, respectively. The interactions have not been correlated with clinical significance in all cases. Diminished end-organ responsiveness may account for reduced drowsiness in smokers receiving chlorpromazine and benzodiazepines, compared with non-smokers. Smoking has been associated with diminished pain tolerance, requiring increased dosages of morphine, pethidine (meperidine) and propoxyphene. Enzyme-inducers such as carbamazepine, phenytoin and phenobarbitone appear to be minimally affected by cigarette smoke, perhaps because hepatic enzymes are already maximally stimulated. Codeine, corticosteroids and nortriptyline do not appear to be affected by cigarette smoke. The bioavailability of glutethimide is higher in smokers, but this has not been associated with greater efficacy. The effect of smoking on paracetamol (acetaminophen) has been variable, depending on the extent of smoking, and does not appear to be of clinical significance.

Role of a bulged A residue in a specific RNA-protein interaction.

The translational operator of the R17 replicase gene contains a bulged A residue that is essential for the specific binding to R17 coat protein. A large number of operator variants have been synthesized to more precisely examine the role of the bulged A residue on this specific protein-RNA interaction. By use of RNA ligase and transcription of synthetic DNA templates by T7 RNA polymerase, 14 different nucleotides were introduced to the bulged A position of three different coat protein binding fragments. The affinity between coat protein and each fragment was determined by a nitrocellulose filter binding assay. The data indicate that while functional groups on N1, C2, C6, N7, and 2'OH of the bulged A can be substituted without greatly changing protein binding, bulky substituents cannot be tolerated at these positions. Data from additional fragments that have base-pair changes adjacent to the bulged A suggest that the propensity of the bulged A to intercalate into the helix can affect protein binding.

Identifying patients who might benefit from free phenytoin monitoring.

Guidelines for measuring free drug concentrations in serum have become necessary due to the easy availability of these assays as a result of the introduction of commercial kits. The present study was performed to identify patients or groups of patients in whom the serum free phenytoin fraction varied from normal, such that they might benefit from measurement of serum free phenytoin. Three hundred fourteen samples submitted for routine phenytoin analysis were studied by enzyme-modified immunoassay technique (EMIT). Thirty-eight patients on phenytoin monotherapy and without other factors thought to affect protein binding of this drug had a mean (+/- SD) free phenytoin fraction of 9.8 +/- 1.8% of total concentration (mean serum albumin concentration 43.4 +/- 3.9 g/L). The free fraction was elevated by administration of comedications which are themselves highly protein bound, and in those patients who were hypoalbuminaemic (serum albumin less than 30 g/L). The groups studied were not mutually exclusive, but stepwise regression analysis showed that other factors known to affect serum albumin (e.g., age greater than 65 years, liver or renal disease, or pregnancy) did not, in themselves, produce a significant effect on free phenytoin fraction. Similarly, an elevated total serum phenytoin concentration was not a significant factor in producing an elevation in free phenytoin fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chemical modifications on the surface- and protein-binding properties of the light chain of human high molecular weight kininogen.

The light chain of kallikrein-cleaved human high molecular weight kininogen is solely responsible for its cofactor activity in blood clotting. Sequencing of the NH2-terminal region of the light chain reported herein identified the third kallikrein cleavage site of high molecular weight kininogen as Arg-437. The co-factor activity of high molecular weight kininogen consists of the capacity to bind to negatively charged surfaces and to factor XI or prekallikrein. Chemical modification of the histidines by either photooxidation or ethoxyformic anhydride affected the equivalent of 14-16 of 23 histidines available and resulted in over 90% loss in procoagulant activity. The modified protein had drastically reduced surface- and zinc-binding capacity, but it bound successfully to either factor XI or prekallikrein. In contrast, modification of two carboxyl groups, which led to approximately 80-90% loss of procoagulant activity, seriously compromised protein binding but left surface binding unaffected. All 3 tryptophans were modified at pH 4.0 with N-bromosuccinimide with a 70% reduction in procoagulant activity, but only 1 tryptophan was available for reaction at pH 7.35, resulting in a 50% loss in activity. Tryptophan modification at acidic pH affected protein binding but did not modify surface or zinc binding. Modification of both available tyrosine and 9 of 18 available lysine residues did not have a significant effect on the procoagulant activity of the light chain. These studies indicate that histidines participate in surface binding and that free carboxyl groups and tryptophan side chains are involved in binding of high molecular weight kininogen to other clotting factors.

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

The effects of menadione (2-methyl-1,4-naphthoquinone) metabolism on intracellular soluble and protein-bound thiols were investigated in freshly isolated rat hepatocytes. Menadione was found to cause a dose-dependent decrease in intracellular glutathione (GSH) level by three different mechanisms: (a) Oxidation of GSH to glutathione disulfide (GSSG) accounted for 75% of the total GSH loss; (b) About 15% of the cellular GSH reacted directly with menadione to produce a GSH-menadione conjugate which, once formed, was excreted by the cells into the medium; (c) A small amount of GSH (~10%) was recovered by reductive treatment of cell protein with NaBH 4, indicating that GSH-protein mixed disulfides were also formed as a result of menadione metabolism. Incubation of hepatocytes with high concentrations of menadione (>200 μ m) also induced a marked decrease in protein sulfhydryl groups; this was due to arylation as well as oxidation. Binding of menadione represented, however, a relatively small fraction of the total loss of cellular sulfhydryl groups, since it was possible to recover about 80% of the protein thiols by reductive treatments which did not affect protein binding. This suggests that the loss of protein sulfhydryl groups, like that of GSH, was mainly a result of oxidative processes occurring within the cell during the metabolism of menadione.

Fluid Shifts and Other Factors Affecting Plasma Protein Binding of Prednisolone by Equilibrium Dialysis

The effects of drug stability, radioactive tracer purity, buffer composition, protein concentration, and fluid shifts on the nonlinear plasma protein binding of prednisolone were examined by equilibrium dialysis. Prednisolone exhibits a concentration-dependent degradation; however, the limited extent of this does not affect protein binding. Impure tritiated prednisolone used as a tracer produces incorrect, low fractional binding values with the binding parameters generated for transcortin affected more than those for albumin. Isotonic sodium phosphate and Krebs original Ringer phosphate buffers yield similar fractional binding of prednisolone and identical protein binding parameters. Fractional binding of the steroid decreases with total plasma protein concentration, but the association constants remain constant over a twofold dilution of plasma proteins. Further dilution increases these parameters. A time-dependent colloidal osmotic fluid shift during dialysis causes dilution of plasma protein concentrations and diminished drug binding. Theoretical simulations show that the osmotic fluid shifts produce the largest changes in fractional binding for compounds that are bound by low-capacity proteins with low association constants (K < 106 M−1). A mathematical equation was developed to correct bound drug concentrations and fraction bound for protein dilution caused by this effect. The fluid shifts can be prevented by the addition of dextran (mol. wt. 70,000) to the dialysis buffer in a concentration of 55% of the total protein concentration. Multiple factors can diminish the nonlinear prednisolone binding as artifacts during equilibrium dialysis, but the changes are relatively modest.

Factors Influencing Serum Protein Binding of Lidocaine in Humans

Several factors that may affect protein binding of lidocaine in human serum were studied in normal volunteers. Evidence was obtained for the presence of two classes of lidocaine binding sites with strikingly different affinity constants (k) and capacities (nP); k1 equals 1.3 x 10(5)M(-1), n1P1 equals 1.7 x 10(-5)M, and k2 equals 6.4 x 10(1)M(-1), n2P2 equals 6.9 x 10(-3)M. The low affinity binding sites are probably on serum albumin, whereas the high affinity site may be located on alpha 1 acid glycoprotein. At a lidocaine serum concentration of approximately 1.4 microgram/ml, it was observed that acidosis (pH 7.4 leads to 7.2) caused lidocaine-free fraction to increase from 0.29 to 0.36 (p less than 0.01) and that the addition of the lidocaine metabolites 3-hydroxylidocaine, 4-hydroxylidocaine, monoethylglycinexylidide, and glycinexylidide had no effect on the binding of lidocaine. Bupivacaine, disopyramide, and quinidine (in concentrations that are observed clinically) caused a significant increase (p less than 0.01) in the free fraction of lidocaine in serum (23%, 21%, and 34%, respectively. Interestingly, N-depropyl disopyramide, dihydroquinidine, procainamide, N-acetyl procainamide, and propranolol had no effect on lidocaine binding.

The effect of disease states on serum protein binding of antimicrobials

Serum protein binding of antimicrobials is altered in various disease states. In uremia, the binding of many organic acids to serum proteins is reduced. The defect in binding is 1. greater than can be accounted for by hypoalbuminemia alone, 2. unchanged by dialysis, 3. transferred in the protein but not the ultrafiltrate fraction, 4. corrected by a successful kidney transplant, and 5. corrected by treatment with activated charcoal at low pH. These data suggest that the binding defect is due to the accumulation of an inhibitor. The nature of the inhibitor is currently unknown. The accumulation of certain endogenous compounds such as bilirubin and free fatty acids (FFA) can also affect protein binding of antimicrobials. Elevation of the molar ratio of FFA to albumin during pump perfusion in patients undergoing cardiac bypass produces a binding defect. Various disease states probably behave similarly.

A Systematic Study of Variables Affecting Protein Binding of Thyroxine and Triiodothyronine in Serum

A Systematic Study of Variables Affecting Protein Binding of Thyroxine and Triiodothyronine in Serum

A Systematic Study of Variables Affecting Protein Binding of Thyroxine and Triiodothyronine in Serum

Using an ultrafiltration and resin dialysis method, the effect of pH, temperature, ionic medium, ionic strength, drugs, dilution, and total hormone concentrations on the free thyroxine (T4) and triiodothyronine (T3) fractions in serum is reported. It is shown that [1] the effect of pH, ionic medium, and tracer dose of T4 is stronger than previously realized; [2] endogenous dialysable competitors cause the so-called paradoxial dilution effect; [3] tris buffer, 0.16 mol/1, with pH 7.35 at 37 °C is a superior dilution medium; and [4] the ratio between T4 and T3 binding constants to thyroxine-binding globulin, thyroxine-binding prealbumin, and albumin are rougly 20, 10, and 6, respectively. This study suggests that the free T4 and T3 fractions in serum will vary in proportion to the various concentrations of total hormones or binding competitors in different states.