Bifunctional small molecules that mediate the degradation of extracellular proteins.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

In vitro detection of allergen sensitized basophils by HSA-DNP antigen-anchored liquid crystal microdroplets.

A simple and easy to handle biosensing technique for in vitro detection of HSA-DNP antigen induced allergen reactions in patients has been developed through the detection of sensitized basophils expressed with anti-IgE receptor (FcεRI) by using human serum albumin-dinitrophenol (HSA-DNP) antigen-anchored liquid crystal (LC) microdroplets emulsion. The radial to bipolar transition in nematic 4-cyano-4'-pentyl biphenyl liquid crystal molecules (5CB) confined in HSA-DNP antigen anchored LC microdroplets (8.5 pg HSA-DNP/LC microdroplet) is found to be sensitive in PBS solution in detection of allergen sensitized basophils expressed with a minimum amount of anti HSA-DNP (anti-IgE) receptor (≥4.5 pg/basophil). The detection of allergen sensitized basophils was possible within a contact time of 30 min in presence of control cells and with 10% solution of human blood plasma. The HSA-DNP antigen anchored LC microdroplets in presence of macrophages or non-sensitized basophils did not show radial to bipolar transition in 5CB molecules in PBS or solution with 10 wt% human blood plasma. Thus HSA-DNP antigen anchored LC microdroplets biosensor may be used for in vivo detection of stage I allergen reaction basophils in blood samples.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones.

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.

Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.

D-amino carboxamide-based recruitment of dinitrophenol antibodies to bacterial surfaces via peptidoglycan remodeling.

During the past few decades there has been a rapid emergence of multidrug resistant bacteria afflicting human patients. At the same time, reduced output from pharmaceutical industry in this area precipitated a sharp decrease in the approval of new antibiotics. The combination of these factors potentially compromises the ability to effectively combat bacterial infections. While traditional drug discovery efforts continue in the pursuit of small molecule agents that disrupt bacterial growth, non-traditional efforts could serve to complement antimicrobial strategies. We recently demonstrated our ability to remodel the surface of bacterial cells using unnatural D-amino acids displaying the antigenic dinitrophenyl (DNP) handle. These immune stimulant D-amino acids derivatives were metabolically incorporated onto the peptidoglycan of bacteria via a promiscuous surface-anchored transpeptidase. The covalent modification of DNP moieties onto the peptidoglycan led to the anti-DNP antibody opsonization of the bacterial cell surface. Herein, we show that the amidation of the C-terminus to generate DNP-displaying D-amino carboxamide drastically improves antibody recruitment. Antibody opsonization using the D-amino carboxamide agent is observed at lower concentrations than the D-amino acid counterpart. In addition, the recruitment of endogenous antibodies in pooled human serum to the DNP-modified bacterial cell surface is demonstrated for the first time. We envision that the C-terminus amidation of DNP-conjugated D-amino acids could potentially facilitate translation of these results to in vivo animal disease models.

Dinitrophenol-Hyaluronan Conjugates as Multivalent Antibody-Recruiting Glycopolymers for Targeted Cancer Immunotherapy.

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG3 -DNP]8 , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG3 -DNP]8 significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

Making Weak Antigens Strong: Modifying Antigens by Dinitrophenol or Arsynyl Coupling.

Many compounds on their own do not have all of the properties needed to induce a strong antibody response. However, small changes in the structure of an antigen can often greatly alter the immunogenicity of a compound. Common methods for doing so include the addition of small modifying groups such as dinitrophenol or arsenate to the molecules. These techniques either alter regions of the immunogen to provide better sites for T-cell binding or expose new epitopes for B-cell binding. The techniques are rapid and easy, and have been used extensively as a general procedure to increase the chances of raising antisera, particularly against well-conserved antigens.

Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis.

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.

Computational models for structure-hydrophobicity relationships of 4-carboxyl-2,6-dinitrophenyl azo hydroxynaphthalenes.

Some phenyl azo hydroxynaphthalene dyes (e.g., sunset yellow) are certified as approved colorants for food, cosmetics, and drug formulations. The hydrophobicity of 4 newly synthesized azo dyes of the phenyl azo hydroxynaphthalene class was investigated, as a training set, with the goal of developing models for quantitative structure-property relationships (QSPR). Retention behavior of the molecules reversed-phase thin-layer chromatography (RPTLC) was investigated using liquid paraffin-coated silica gel as the stationary phase. Mobile phases consisted of aqueous mixtures of methanol, acetone, and dimethylformamide (DMF). Basic hydrophobicity parameter (Rmw), specific hydrophobic surface area (S), and isocratic chromatographic hydrophobicity index (phio) were computed from the chromatographic data. The hydrophobicity index (Rm) decreased linearly with increasing concentration of organic modifiers. Extrapolated Rmw values obtained by using DMF and acetone differ significantly from the value obtained by using methanol as organic modifier [P < 0.05, 1-way analysis of variance (ANOVA), Tukey's multiple comparison test]. Structure-property relationships showed that hydrophobicity was dependent on type and position of naphthalene ring substituents. Rm decreased with the presence of a highly polar substituent (e.g., COOH). Owing to intramolecular interaction, Rm increased when the common hydroxyl group (OH) is positioned ortho to the azo group, relative to para positioning, in 2 positional isomers. Pattern recognition data analysis underscores the utility of phio as a more accurate hydrophobicity descriptor than Rmw. Phio is negatively correlated with theoretically calculated density, surface tension, and refractive index for the molecules. These models could be used to predict toxicity (absorption, distribution, metabolism, excretion, toxicity; ADMET) properties of the azo dyes and may also play useful roles in computer-assisted molecular discovery of nontoxic azo dyes.

In Silico Design, Synthesis and Bioactivity of N-(2, 4-Dinitrophenyl)-3-oxo- 3-phenyl-N-(aryl) Phenyl Propanamide Derivatives as Breast Cancer Inhibitors.

BACKGROUND: Breast cancer is a systemic disease which has challenged physicians worldwide as it is the most predominant cancer in women often leading to fatality. One of the types of treatment is chemotherapy which includes targeted oral or intravenous cancer-killing drugs. Treatment options are often limited to surgery and/or chemotherapy. OBJECTIVE: The discovery and design of new small molecule estrogen inhibitors is necessitated in order to circumvent the problem of drug-induced resistance in chemotherapy resulting in disease relapse. Chemoinformatics facilitates the design, selection and synthesis of new drug candidates for breast cancer by providing efficient in silico techniques for prediction of favourable ADMET properties, and structural descriptors to profile druggability of a compound. METHOD: Several molecules selected from docking studies were synthesized and evaluated for their biological activities on the MCF-7 (human breast cancer) cell line. RESULTS: These estrogen inhibitors displayed good inhibitory activity with high selectivity and hence can be further progressed as drug candidates effective against breast cancer. CONCLUSION: It is for the first time that N-(2, 4-dinitrophenyl)-3-oxo-3-phenyl-N-(aryl) phenylpropanamide derivatives were reported to be biological active as potential breast cancer inhibitors.

Preparation and identification of anti-2, 4-dinitrophenyl monoclonal antibodies.

2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)).

Targeting dinitrophenol to mitochondria: limitations to the development of a self-limiting mitochondrial protonophore.

The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

Non-Faradaic Electrochemical Detection of Exocytosis from Mast and Chromaffin Cells Using Floating-Gate MOS Transistors.

We present non-faradaic electrochemical recordings of exocytosis from populations of mast and chromaffin cells using chemoreceptive neuron MOS (CνMOS) transistors. In comparison to previous cell-FET-biosensors, the CνMOS features control (CG), sensing (SG) and floating gates (FG), allows the quiescent point to be independently controlled, is CMOS compatible and physically isolates the transistor channel from the electrolyte for stable long-term recordings. We measured exocytosis from RBL-2H3 mast cells sensitized by IgE (bound to high-affinity surface receptors FcεRI) and stimulated using the antigen DNP-BSA. Quasi-static I-V measurements reflected a slow shift in surface potential () which was dependent on extracellular calcium ([Ca]o) and buffer strength, which suggests sensitivity to protons released during exocytosis. Fluorescent imaging of dextran-labeled vesicle release showed evidence of a similar time course, while un-sensitized cells showed no response to stimulation. Transient recordings revealed fluctuations with a rapid rise and slow decay. Chromaffin cells stimulated with high KCl showed both slow shifts and extracellular action potentials exhibiting biphasic and inverted capacitive waveforms, indicative of varying ion-channel distributions across the cell-transistor junction. Our approach presents a facile method to simultaneously monitor exocytosis and ion channel activity with high temporal sensitivity without the need for redox chemistry.

The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness.

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross-reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4-dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross-reactants are themselves bound specifically-close derivatives of these cross-reactants show very low or no binding to SPE7. It has been suggested that cross-reactivity is simply due to "hydrophobic stickiness", nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross-reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1-8. By comparing the sequences and binding patterns of SPE7 and B1-8, we address the relationship between affinity maturation, specificity, and cross-reactivity.

Inhibition by dexamethasone of interleukin 13 production via glucocorticoid receptor-mediated inhibition of c-Jun phosphorylation.

The antigen stimulation of RBL-2H3 cells induced interleukin 13 (IL-13) production, which was inhibited by the steroidal anti-inflammatory drug dexamethasone and by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Dexamethasone did not inhibit the antigen-induced phosphorylation of JNK but inhibited that of c-Jun. In a cell-free system, the phosphorylation of glutathione S-transferase-fused c-Jun by recombinant JNK was not inhibited by dexamethasone but was inhibited by the addition of recombinant glucocorticoid receptor (GR). These findings suggest that the inhibition of antigen-induced IL-13 production by dexamethasone is due to the GR-mediated inhibition of c-Jun phosphorylation induced by JNK.

A tracer study with systemically and locally administered dinitrophenylated osteopontin.

Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a "surgical window" in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected.

Direct interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c oxidoreductases probed by sensitivity to quinone-related inhibitors.

The electron-transfer activities of bovine heart mitochondrial complexes I, II, and III, but not complex IV, were simultaneously inhibited by 2-alkyl-4,6-dinitrophenols to a different extent. The extent of inhibition of NADH and succinate oxidase activities by dinitrophenols was compared with that of individual complex activities using submitochondrial particles. The extent of inhibition of succinate oxidase activity by 1-methylpropyl and 1-methylbutyl derivatives was much larger than that of NADH oxidase activity. This large inhibition of succinate oxidase activity seemed not to be explainable by the extent of inhibition of individual complex activities (i.e., complexes II and III activities), based upon the homogeneous ubiquinone pool model. On the other hand, other dinitrophenols (n-propyl, 1-methylpentyl, 1-methylhexyl, and tert-butyl derivatives) very similar to the above compounds did not elicit such anomalous inhibitory action, indicating that the action of 1-methylpropyl and 1-methylbutyl derivatives is highly specific to their structure. The anomalous inhibition by these two compounds was also observed with the isolated succinate-cytochrome c oxidoreductase, in which there is no ubiquinone pool behavior [Rich, P.R. (1984) Biochim. Biophys. Acta 768, 53-79]. However, when the succinate-cytochrome c reductase of which the activity had been partially restored by adding phospholipid and exogenous quinone to the phospholipid- and ubiquinone-depleted succinate-cytochrome c reductase was assayed, the anomalous inhibitory action of interest was undetectable. These results indicated that electron-transfer between complexes II and III, which is mediated not only by free-form, but also by protein-bound ubiquinone, occurs in the mitochondrial membrane. The fact that the anomalous inhibition of succinate oxidase activity of submitochondrial particles was sensitive to changes in the external osmotic pressure which affected the total area of the particle supports this notion.

Dinitrophenol derivatization of proteolytic products and its application in the assay of protease(s) activity.

A spectrophotometric method based on dinitrophenol (DNP) derivatization of proteolytic products was developed for monitoring the increase in NH(2)-groups as a function of protease activity. DNP derivatization of amino acids and proteolytic products was carried out at an alkaline pH of 8.8, in presence of 2,4-dinitrofluorobenzene (DNFB), followed by the stabilization of products by adjusting the pH to approximately 2.5. Using casein as substrate, under the defined assay conditions for proteases, trichloroacetic acid soluble proteolytic products were derivatized with DNFB reagent. Though alkaline pH favored the DNP derivatization of primary amino compounds, the products formed were found to be unstable. However, upon adjusting the pH to 2.5+/-0.1, DNP derivatives of amino acids and proteolytic products were found to be stable with identical lambda(max) of 395 nm. The utility of the method was evaluated by assaying the proteolytic activities of trypsin and calcium activated neutral protease (CANP). Proteolytic activity was quantified by employing the molar extinction coefficient of DNP derivatives of an equimolar concentration of glutamate and glycine. By employing this method, CANP activity in different regions of rat brain was determined. The proposed method to monitor the increase in NH(2)-end groups as a function of proteolytic activity could be employed to assay the activity of proteases.

Effect of the weakly acidic uncoupler 2,4-dinitrophenol and dimethyl sulfoxide on the coordination of Mg2+ with ATP. Possible mechanism of activation of the isolated F1-ATPase by 2,4-dinitrophenol.

The exchange rate constants between Mg2(+)-free and Mg2(+)-bound ATP were determined under various conditions by line shape analysis of the 31P-NMR spectrum based on the exchange reaction, and the thermodynamic parameters of this exchange reaction were determined from the temperature dependence of its rate constants. Analysis of the activation enthalpy change delta H showed that Mg2+ is coordinated with the beta- and gamma-phosphoryl groups of ATP asymmetrically, being in closer proximity to the beta-phosphoryl group. The weakly acidic uncoupler 2,4-dinitrophenol increased this asymmetric coordination of Mg2+, and this effect was enhanced by the further addition of dimethyl sulfoxide. The hydrolysis of ATP in aqueous solution correlated well with the degree of asymmetry of Mg2+ coordination. Thus, this asymmetric coordination specifically weakens the O-P gamma bond at which specific cleavage of ATP catalyzed by most ATPases takes place in the presence of Mg2+. In this paper, the mechanism of activation of isolated ATPase (F1-ATPase) by 2,4-dinitrophenol, and that of ATP synthesis by isolated F1-ATPase in the presence of dimethyl sulfoxide are considered on the basis of these results. The essential role of the OH group of Ser-174 of the beta-subunit of F1-ATPase in ATP hydrolysis is also discussed.

Biodegradable microspheres. XIII: Immune response to the DNP hapten conjugated to polyacryl starch microparticles.

Polyacryl starch microparticles are being investigated for use as drug carriers, especially in the treatment of intracellular parasitic diseases. The purpose of this work was to investigate the possible immune response to drugs coupled to the microparticles, using the dinitrophenyl group (DNP) as a model. The humoral immunogenicity of DNP hapten-conjugated polyacryl starch microparticles has been examined in mice. Microparticle:hapten complexes with different biodegradability were tested, as well as different dosages and routes of administration. Two DNP derivatives, Lys(DNP) and Leu-Ala-Lys(DNP), were coupled to microparticles. It was shown that Lys(DNP) was released from the particles by lysosomal enzymes only from the conjugate with the tripeptide DNP derivative. The DNP conjugated to the particles via the biodegradable Leu-Ala-Lys arm induced only a weak immune response without memory. No response was detected after injection of the Lys(DNP) microparticles. Thus, there should be no major immunological obstacles in using drug:microparticle complexes in the treatment of, for example, parasitic diseases.