MRP4/ABCC4 As a New Therapeutic Target: Meta-Analysis to Determine cAMP Binding Sites as a Tool for Drug Design

MRP4 transports multiple endogenous and exogenous substances and is critical not only for detoxification but also in the homeostasis of several signaling molecules. Its dysregulation has been reported in numerous pathological disorders, thus MRP4 appears as an attractive therapeutic target. However, the efficacy of MRP4 inhibitors is still controversial. The design of specific pharmacological agents with the ability to selectively modulate the activity of this transporter or modify its affinity to certain substrates represents a challenge in current medicine and chemical biology. The first step in the long process of drug rational design is to identify the therapeutic target and characterize the mechanism by which it affects the given pathology. In order to develop a pharmacological agent with high specific activity, the second step is to systematically study the structure of the target and identify all the possible binding sites. Using available homology models and mutagenesis assays, in this review we recapitulate the up-to-date knowledge about MRP structure and aligned amino acid sequences to identify the candidate MRP4 residues where cyclic nucleotides bind. We have also listed the most relevant MRP inhibitors studied to date, considering drug safety and specificity for MRP4 in particular. This meta-analysis platform may serve as a basis for the future development of inhibitors of MRP4 cAMP specific transport.

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Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

10.1055/s-0039-1684551 ◽

1979 ◽

Author(s):

B. Kudryk ◽

M. Blombäck

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Specific Activity ◽

High Specific Activity ◽

Antigenic Determinants ◽

Affinity Binding ◽

Compact Structure ◽

High Affinity Binding ◽

High Affinity ◽

Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

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The Diageotropica Mutant of Tomato Lacks High Specific Activity Auxin Binding Sites

Science ◽

10.1126/science.245.4913.52 ◽

1989 ◽

Vol 245 (4913) ◽

pp. 52-54 ◽

Cited By ~ 55

Author(s):

G. R. HICKS ◽

D. L. RAYLE ◽

T. L. LOMAX

Keyword(s):

Binding Sites ◽

Specific Activity ◽

High Specific Activity ◽

Auxin Binding

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THE DEVELOPMENT OF A RADIOIMMUNOASSAY FOR ARGININE-VASOPRESSIN: PRODUCTION OF ANTISERA AND LABELLED HORMONE; SEPARATION TECHNIQUES; SPECIFICITY AND SENSITIVITY OF THE ASSAY IN AQUEOUS SOLUTION

Journal of Endocrinology ◽

10.1677/joe.0.0520279 ◽

1972 ◽

Vol 52 (2) ◽

pp. 279-288 ◽

Cited By ~ 18

Author(s):

C. R. W. EDWARDS ◽

T. CHARD ◽

MURIEL J. KITAU ◽

MARY L. FORSLING ◽

J. LANDON

Keyword(s):

Arginine Vasopressin ◽

Specific Activity ◽

Limit Of Detection ◽

High Specific Activity ◽

Ion Exchange Resin ◽

Cross Reactivity ◽

Amino Acid Sequences ◽

Simple Method ◽

Specificity And Sensitivity ◽

Chloramine T

SUMMARY A radioimmunoassay for vasopressin was developed using antibodies produced against conjugated and non-conjugated arginine vasopressin. Despite the fact that the vasopressin molecule has only eight amino acids, cross reactivity studies showed that these antibodies were specific for different amino acid sequences. Labelled hormone of high specific activity (350–800 μCi/μg) was produced by a modification of the chloramine-T method. Unreacted iodide was removed by the batchwise addition of an ion-exchange resin. Other techniques of purification produced no advantage over this simple method. Several methods of separating antibody-bound and free hormone were studied. All except chromatoelectrophoresis proved satisfactory. Ammonium sulphate or ethanol precipitation of bound hormone was chosen because of simplicity, speed and reproducibility. The lower limit of detection of the assay was 80 pg arginine vasopressin/ml diluent buffer. Therefore an extraction and concentration procedure is necessary for the measurement of basal circulating levels of the hormone.

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Fragment D Immunoreactivity: The Influence of Iodination and Calcium Environment

10.1055/s-0038-1665823 ◽

1979 ◽

Author(s):

B. Kudryfc ◽

N. Blombäck

Keyword(s):

Conformational Changes ◽

Specific Antibody ◽

Binding Sites ◽

Specific Activity ◽

High Specific Activity ◽

The Other ◽

Antigenic Determinants ◽

Affinity Binding ◽

Compact Structure ◽

Chloramine T

Human fragment D (Fg-Ds) has been iodinated using both the Chloramine-T and lactoperoxidase methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40%, the other labeled product bound up to 85%. A correlation between the degree of immunoreactivity and avidity for a fibrinmonomer conjugate was found also. Fibrinmonomer bound about twice the amount of lactoperoxidase iodinated Fg-Ds as it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. affinity binding sites for calcium have recently been demonstrated in fibrinogen. The presence of bound calcium is also believed to protect Fg-Ds from further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that some modification of antigenic determinants takes place as a consequence of calcium in the environment.

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Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

Applied and Environmental Microbiology ◽

10.1128/aem.03523-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1561-1569 ◽

Cited By ~ 50

Author(s):

Linhua Huang ◽

Yu Liu ◽

Yan Sun ◽

Qiaojuan Yan ◽

Zhengqiang Jiang

Keyword(s):

Amino Acid ◽

Biochemical Characterization ◽

Specific Activity ◽

High Specific Activity ◽

Rhizomucor Miehei ◽

Drug Dosage ◽

Amino Acid Sequences ◽

Content Type ◽

Leukemia Treatment ◽

Glutaminase Activity

ABSTRACTA novel fungal gene encoding theRhizomucor mieheil-asparaginase (RmAsnase) was cloned and expressed inEscherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reportedl-asparaginases. The purifiedl-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, thisl-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

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Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657178 ◽

1982 ◽

Vol 47 (03) ◽

pp. 244-248 ◽

Cited By ~ 47

Author(s):

D P Thomas ◽

Rosemary E Merton ◽

T W Barrowcliffe ◽

L Thunberg ◽

U Lindahl

Keyword(s):

Antithrombin Iii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Blood Levels ◽

Thrombin Time ◽

High Affinity ◽

Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655440 ◽

1962 ◽

Vol 08 (03) ◽

pp. 425-433 ◽

Cited By ~ 1

Author(s):

Ewa Marciniak ◽

Edmond R Cole ◽

Walter H Seegers

Keyword(s):

Snake Venom ◽

Thrombolytic Agent ◽

Sodium Citrate ◽

Specific Activity ◽

High Specific Activity ◽

Citrate Solution ◽

Clotting Factors ◽

Activation Products ◽

Russell’S Viper ◽

Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

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