Lucia Christodoulides

Studies of the chemistry of the luminal plasma membrane of rat bladder epithelial cells

Abstract The lipid and protein components of the luminal plasma membrane of the rat bladder have been analysed. The principal lipid components are cholesterol, phosphatidylcholine, phosphatidylethanolamine and cerebroside. Saponification showed the presence in the phospholipids of palmitic, stearic, oleic, linoleic, linolenic, eicosatrienoic and arachidonic acyl groups. A range of proteins was separated according to isoelectric point and polypeptide molecular weight. The amino acid composition of the luminal plasma membrane protein differed from that of other cell subfractions in having an unusually high proline content. These factors are discussed in relation to both the unusual ultrastructure of the membrane and its low permeability to water and electrolytes.

Isolation, properties and tissue distribution of rat glutathione transferase E

Glutathione transferase E (5‐5) Rat liver Purification Subunit size Tissue distribution

Characterization and purification of fatty acid-binding protein in rat and human adipose tissue.

A protein with properties similar to fatty acid-binding protein has been isolated from rat and human adipose tissue. Comparison of fatty acid-binding protein from rat liver and adipose tissue and human adipose tissue shows that all have approximately similar molecular weights. Immunologically, rat liver fatty acid-binding protein is similar to the protein characterized from rat adipose tissue. In isolated rat fat cells the fatty acid-binding protein was demonstrated to be involved in the uptake and esterification of long-chain fatty acids. These observations constitute evidence for a potential role of this protein in the fatty acid metabolism of adipocytes.

Two specific azodye-carcinogen-binding proteins of the rat liver: The identity of amino acid residues which bind the azodye

Abstract Two soluble liver proteins in the rat have a high affinity of binding for metabolites of azodye carcinogens. One protein is referred to as the basic azodye-binding protein (mol. wt. 45 000, pI 8.4) and the other as the small molecular weight protein (mol. wt. 13 800). A study has been made of the amino acid residues which bind the azodye metabolites in these two proteins. Azodye-bound components were released from these proteins by Pronase digestion followed by refluxing in alkali-ethanol. They were then separated by ‘fingerprinting’. 35S-Labelled amino acids were administered to rats together with azodye. After the administration of L -[35S]methionine, azodye-bound components from both types of protein were radioactive: after the administration L -[35S]cysteine, azodye components from the basic protein only were radioactive. Thus, since methionine is an essential amino acid, in the basic azodye-binding protein azodye metabolites bind to cysteinyl residues and in the small molecular weight azodye-binding protein, binding is to methionyl residues.

Inhibition of binding of benzo(a)pyrene metabolites to nuclear DNA by glutathione and glutathione S-transferase B

Abstract Incubation of benzo(a)pyrene with the hepatic nuclear fraction isolated from 3-methylcholanthrene-treated rats resulted in the binding of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene to nuclear DNA. Addition of cytosol to the incubation decreased the level of DNA-bound metabolites especially those derived from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. This inhibitory effect of cytosol was related to its content of reduced glutathione and could be reproduced by the replacement of cytosol by glutathione and glutathione S-transferase B. These results suggest that the level of glutathione and glutathione S-transferases may be of prime importance in regulating the accumulation of reactive intermediates of benzo(a)pyrene in the intact cell.

Purification and characterization of fatty acid binding protein in mammalian lung

A fatty acid-binding protein (FABP) has been isolated and characterized from rat lung tissue. Rat lung FABP has a slightly higher molecular weight than liver FABP, but immunologically, lung FABP is similar to that of liver FABP. Long chain acyl CoA synthetase, a key enzyme in fatty acid metabolism is stimulated by partially purified lung FABP, suggesting a physiologic role of the protein in the activation of long chain fatty acids in pulmonary tissue.

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.

The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies.

Purified glutathione(GSH)-S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEt3). With 1-chloro-2,4-dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10(-3) M the inhibition constants for transferases A and C at 25 degrees C are similar and very low, 3.2 . 10(-8) M and 5.6 . 10(-8) M respectively, whereas for transferase B the inhibition constant is 3.5 . 10(-5) M. Equilibrium-dialysis experiments carried out at 4 degrees C in the absence of GSH give apparent dissociation constants of 7.1 . 10(-4) M and 3.4 . 10(-4) M for transferases A and B respectively, but if 5 . 10(-3) M glutathione is included in the dialysis solutions these values fall to 2.0 . 10(-7) M and 2.6 . 10(-5) M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSH-SnEt3, in which GSH and SnEt3 may or may not interact directly; or are possibly quaternary complexes, enzyme-(GSH)2-SnEt3. SnEt3 could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases.

Mercapturic acid biosynthesis: The separate identities of glutathione-S-aryl chloride transferase and ligandin

Summary Glutathione-S-aryl chloride transferase (E.C.2.5.1.13) and ligandin have similar molecular weights, but are different electrophoretically and immunologically and can be separated by isoelectric focusing. Glutathione-S-aryl chloride transferase has been obtained as a by-product of ligandin purification. The different roles of glutathione-S-aryl chloride transferase and ligandin in excretion by the mercapturic acid pathway are discussed.