Rat Kidney Cytosol Research Articles

The pathogenesis of pseudohyperaldosteronism from carbenoxolone.

Carbenoxolone is a derivative of glycyrrhetinic acid used for the treatment of peptic ulcer and gastritis, with salt and water retention a very common side-effect. To investigate this drug-induced pseudohyperaldosteronism we have studied 6 male volunteers before, during and after treatment with carbenoxolone for 7 days. Serum, urinary and sweat electrolytes values were consistent with a mineralocorticoid-like effect of drug administration. PRA was suppressed, and plasma cortisol and aldosterone progressively decreased over treatment. We have also determined by radioreceptor assay the plasma levels of factors which bind to mineralocorticoid receptors in rat kidney cytosol. The levels of these factors were decreased significantly at day 3 of treatment, suggesting a local renal effect of carbenoxolone to amplify endogenous steroid action. At day 7 the radioreceptor assay values were still decreased but significantly higher than at day 3, suggesting in addition a direct mineralocorticoid effect of the drug. We conclude that the drug is initially effective by amplifying the effect of endogenous steroids, and then when the plasma concentrations of the drug or its metabolites reach a higher plasma concentration, there may also be in addition a direct mineralocorticoid-like effect.

Regulation of aldosterone receptor in rat kidney cytosol by atrial natriuretic factor.

We investigated the effect of atrial natriuretic factor (ANF) on aldosterone receptors in the kidney cytosol, because the binding of aldosterone to aldosterone receptors in the cytosol is considered a critical step of its action. Rat atriopeptin III was injected into male Sprague-Dawley rats (200-250 g) via the femoral vein while under pentobarbital anesthesia, and aldosterone receptors in the kidney cytosol were determined. The maximum binding capacity and dissociation constant were calculated by the Scatchard analysis. Maximum binding capacity of both types of aldosterone receptor (Type I, high affinity and low binding capacity and Type II, low affinity and high binding capacity) gradually decreased after ANF injection, reached the lowest level after 2 hours, and then slightly recovered. When more than 2.5 micrograms/kg of rat atriopeptin III was injected, the density of aldosterone receptors significantly decreased. Injection of 12.5 micrograms/kg of rat atriopeptin III decreased maximum binding capacity of Type I receptor from 42.3 +/- 2.4 (mean +/- SD, n = 6) to 22.8 +/- 3.2 femtomole/mg protein (n = 6) (p less than 0.01), and that of Type II receptor decreased from 388 +/- 46 to 285 +/- 30 fmol/mg protein (p less than 0.01). Dissociation constant of both types of receptors did not change significantly after ANF injection. Plasma aldosterone concentration showed no significant change after ANF injection, and a significant change was noted after ANF administration on aldosterone receptors in the experiments on adrenalectomized rats 7 days after operation. Furosemide had no significant effect on aldosterone receptors in both normal and adrenalectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine conjugate β-lyase of rat kidney cytosol: Characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity

Cysteine conjugate β-lyase (β-lyase) was purified to electrophoretic homogeneity from the kidney cytosol of male Wistar rats. The highly purified enzyme exhibited a monomeric molecular weight of 50,000 Da and was active in the α-β elimination of cysteine conjugates including S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC), and S-(2-benzothiazolyl)- l-cysteine, particularly toward DCVC and TFEC. The purified enzyme also exhibited glutamine transaminase K activity with phenylalanine and α-keto-γ-methiolbutyrate as substrates. An antibody was raised to the purified rat protein in sheep and the crude immune serum affinity purified, yielding a specific antibody that recognized only the β-lyase protein in whole kidney homogenates. Immunocytochemical studies on rat kidney sections stained with the purified antibody revealed that the cytosolic β-lyase enzyme was mainly localized in the pars recta of the proximal tubule in untreated rats. This localization is coincident with the site-specific kidney necrosis produced by hexachloro-1,3-butadiene (HCBD). These results indicate that the tissue localization of β-lyase in the proximal tubule plays an important role in determining the specific nephrotoxicity produced by halogenated alkenes such as HCBD.

Chemical forms of nickel in rat plasma, kidney cytosol and urine after administration of 63NiCl2.

The binding of 63Ni to low-molecular-weight constituents of rat plasma, kidney cytosol and urine has been investigated in vivo and in vitro. Comparative tissue distribution and excretion of 63Ni in rats after administration of 63NiCl2 through various routes revealed that among rat organs, the kidney showed the highest affinity for 63Ni and that radio-nickel was excreted easily into the urine by way of the kidney. Sephadex G-25 and G-200 gel chromatographies of plasma, kidney cytosol and urine after iv or po administration of 63NiCl2 demonstrated that a major component of nickel metabolite in the rat urine may originate from one of three Ni-complexes with low molecular weight in the plasma and kidney cytosol. The behaviors of Ni-complex on thin layer chromatography and Sephadex G-25 gel chromatography suggested that one of the metabolites in the rat urine after iv or po administration of 63NiCl2 existed as a complex of nickel with a picrate-forming substance resembling creatine-related substances.

Use of monolayers of primary rat kidney cortex cells for nephrotoxicity studies

Kidney cells were isolated from rat kidney cortex and maintained in short-term monolayer cultures. A number of important parameters were studied in order to establish the usefulness of these cells for toxicity studies. Despite morphological differences between the cultured cells and similar cells in vivo, many relevant enzyme systems remained present and functional. Intracellular glutathione levels were stable up to 5 days in culture. The glutathione S-transferase activity during culture remained stable although at a lower level than in freshly isolated cells. Whereas rat kidney cytosol contained subunits 4, 7, 2 and 1, 3- and 5-day-old cultures contained glutathione transferase subunits 7, 2 and a small amount of subunit 1. Cytochrome P-450, although measurable in microsomes from freshly isolated cells, could not be determined after 1 day in culture. Organic anion transporters on the basolateral side and γ-glutamyl transpeptidase on the apical side were present. Through cytotoxicity studies, β-lyase activity could be demonstrated in the culture. Hence this monolayer culture system, which can be used in combination with filters, seems to be suitable for studying various mechanisms of nephrotoxicity.

Deacetylation and further metabolism of the mercapturic acid of hexachloro-1,3-butadiene by rat kidney cytosol in vitro.

Hexachloro-1,3-butadiene (HCBD) is more nephrotoxic to female than male rats. Metabolism of HCBD involves conjugation with glutathione followed by formation of the cysteine conjugate S-(pentachloro-1,3-butadienyl) cysteine (PCBD-CYS) and then the mercapturic acid N-acetyl-S-pentachloro-1,3-butadienyl-cysteine (PCBD-NAC). PCBD-NAC is also more nephrotoxic to female rats than male rats. The deacetylation of [14C]-PCBD-NAC to PCBD-CYS and the binding of radiolabelled metabolites to protein has been studied using renal cytosol preparations from male and female rats in vitro, since a sex-related difference in these reactions could explain the difference in nephrotoxicity found in vivo. PCBD-NAC was rapidly metabolised by renal cytosol. The rate of metabolism was similar with either male or female renal cytosol, and the major metabolite identified was PCBD-CYS. N-Acetylation of PCBD-CYS to PCBD-NAC was not detected in the presence of either male or female renal cytosol. Covalent binding of radioactivity from [14C]-PCBD-NAC to cytosolic protein could be detected after 5 min incubation, and although the extent of binding was similar for both male and female cytosol at early time periods, after 60 min incubation more binding was found in the presence of male cytosol. Covalent binding was largely prevented by aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, suggesting a role for this enzyme in the activation of HCBD. These results indicate that the sex differences in the nephrotoxicity of HCBD and PCBD-NAC in the rat are not attributable to differences in the rate of deacetylation of PCBD-NAC to give the proximate nephrotoxin PCBD-CYS.

Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum.

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three 'affinity families' of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.

Enzymatic transformation of mercapturic acids derived from halogenated alkenes to reactive and mutagenic intermediates

The metabolism of the mercapturic acids S-pentachlorobutadienyl- N-acetylcysteine (N-Ac-PCBC), S-trichlorovinyl- N-acetylcysteine(N-Ac-TCVC)andS-dichlorovinyl- N-acetylcysteine (N-Ac-DCVC) by subcellular fractions from male rat liver and kidney homogenates was studied. As a model compound, N-Ac-PCBC, 14C labelled, was synthesised. It was intensively metabolised by cytosolic but not by microsomal enzymes from rat liver and kidney. The major metabolite identified by GC/MS was pentachlorobutadienylcysteine, the amount produced being highest in kidney cytosol. Metabolic conversion of 14C-N-Ac-PCBC by kidney and liver cytosol resulted in covalent binding of radioactivity to protein, binding was strongly inhibited by the β-lyase inhibitor aminooxyacetic add (AOAA). N-Ac-TCVC and N-Ac-DCVC were also transformed by cytosolic enzymes to the corresponding cysteine conjugates (trichlorovinylcysteine and dichlorovinylcysteine). The three mercapturic acids tested were strong mutagens in the Ames-test after addition of rat kidney cytosol. In the absence of cytosol, N-Ac-TCVC and N-Ac-DCVC were weakly but definitely mutagenic, whereas N-Ac-PCBC was not. In contrast to N-Ac-PCBC, the “direct” mutagens N-Ac-TCVC and N-Ac-DCVC were both transformed to pyruvate by bacterial ( S typhimurium TA100) homogenate 100,000 g supernatants. It is concluded that mercapturic acids are deacetylated to the corresponding cysteine conjugates by cytosolic (N-Ac-PCBC, N-Ac-TCVC and N-Ac-DCVC) and bacterial enzymes (N-Ac-TCVC and N-Ac-DCVC) and further cleaved to reactive and mutagenic intermediates by mammalian and/or bacterial β-lyase. The observed activation mechanisms for the mercapturic acids, whose formation from hexa-chlorobutadiene, tetrachloroethylene and trichloroethylene has been proven, might contribute to the nephrotoxicity and nephrocarcinogenicity of the parent alkenes.

Purification and properties of methyl sulfoxide reductases from rat kidney

Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 ± 1000 for methyl sulfoxide reductase I and 24,000 ± 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective K m values of p-bromophenyl-methyl sulfoxide were 2.75 and 1.30 m m for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anionexchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 μ m and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [ 14C]palmitate binding to the protein.

Evidence for the presence of active and inactive forms of cytosolic triiodothyronine binding protein in rat kidney: cooperative action of Ca2+ in NADPH activation.

The effect of NADPH and Ca2+ on 3, 5, 3'-L-triiodothyronine (T3) binding to cytosolic T3 binding protein (CTBP) in rat kidney was investigated in vitro. Extraction of rat kidney cytosol with 10% charcoal at 4 degrees C for 30 min. inactivated specific T3 binding. The decreased T3 binding activity in extracted cytosol could be restored by adding NADPH or Ca2+ to the incubation medium. Each substance increased the capacity for T3 without changes in the affinity for T3. The T3 binding was maximally increased by 25 microM NADPH or by 1.0 mM Ca2+ in the presence of 0.1 mM EDTA. The increase in T3 binding, induced by 25 microM NADPH, was enhanced by adding 0.2-1.0 mM Ca2+ in a concentration dependent-manner. The increase in T3 binding, induced by 1.0 mM Ca2+, was also enhanced by adding 3.125-25.0 microM NADPH. The NADPH-induced increase in T3 binding capacity was amplified by Ca2+ in a multiplicative manner. The results suggested that Ca2+ cooperatively augmented an NADPH function in cytosolic T3 binding.

A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K.

Cysteine conjugate beta-lyase has been purified from rat kidney cytosol. The enzyme is a 100,000-dalton dimer of two 55,000-dalton subunits and has an absorption maximum at 432 nm. The enzyme has phenylalanine alpha-keto-gamma-methiolbutyrate transaminase activity and appears to be identical to rat kidney cytosolic glutamine transaminase K. Metabolism of S-1,2-dichlorovinyl-L-cysteine (DCVC) by the purified enzyme was dependent on the presence of either alpha-keto-gamma-methiolbutyrate or a protein factor which is present in the cytosolic fraction of rat kidney cortex. The protein factor was identified as a flavin containing L-amino acid oxidase which oxidized DCVC to S-(1,2-dichlorovinyl)-3-mercapto-2-oxopropionic acid. S-(1,2-Dichlorovinyl)-3-mercapto-2-oxopropionic acid has not been previously reported as a metabolite of DCVC. The data also show that rat kidney cytosolic glutamine transaminase K catalyzes both a beta-elimination and a transamination reaction with DCVC when alpha-keto-gamma-methiolbutyrate is present and that amino acid oxidase and alpha-keto-gamma-methiolbutyrate stimulate the enzyme activity by providing amino acceptors. When incubations were done with DCVC as substrate in the presence of excess alpha-keto-gamma-methiolbutyrate, the beta-lyase catalyzed beta-elimination and transamination in a ratio of 1:1.3, respectively. Under conditions where most of the alpha-keto-gamma-methiolbutyrate was consumed, the beta-elimination predominated indicating that the S-1,2-dichlorovinyl-3-mercapto-2-oxopropionic acid pool was consumed by transamination after the alpha-keto-gamma-methiolbutyrate had been depleted. The data are discussed with regard to the importance of these pathways as regulators or participants in the toxicity of S-cysteine conjugates.

Active and inactive forms of 3,5,3'-triiodo-L-thyronine (T3)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T3 binding.

Extraction of rat kidney cytosol with 10% charcoal at 4 C inactivated specific T3 binding. The decreased T3 binding in extracted cytosol could be restored by addition of boiled kidney cytosol. Three different factors (a, b, and c) which could increase T3 binding were identified by Sephadex G-50 column chromatography of boiled cytosol. Two factors (b and c) were eluted as relatively small molecules. Factor a was present in small amounts. Factor c was neutralized by incubation with EDTA, but factor b was not. Factor b was not destroyed by trypsin, protease, DNase, or RNase, but was destroyed by alkaline phosphatase. Factor b was destroyed by incubation with nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase in the presence of oxidized glutathione. Although T3 binding to charcoal-extracted cytosol protein was not influenced by reduced glutathione or dithiothreitol, it was markedly increased by NADPH. Maximal activation induced by 50 microM NADPH was not further increased by further addition of endogenous factor b. The elution position of NADPH in gel chromatography corresponded to the elution position of factor b. Factor b or NADPH increased maximal binding capacity without changes in affinity constant. These observations suggest that T3-binding protein in cytosol is present in inactive and active forms and that the active form is generated by NADPH, which is present as one of the activators in cytosol. The effect of these cytosolic T3-binding proteins on nuclear T3 binding in vitro was also studied. In the absence of cytosolic T3-binding protein, [125I]T3 binding to nuclear receptor was decreased by unlabeled T3 in a concentration-dependent manner. In the presence of inactive form of cytosolic T3-binding protein, nuclear [125I]T3 binding was slightly diminished. In the presence of NADPH and cytosolic T3-binding protein, however, the amount of [125I]T3 bound to nuclei markedly decreased, which was associated with an increase of cytosolic [125I]T3 binding. NADPH alone did not influence nuclear T3 binding. These results suggest that T3 binding to nuclear receptor is regulated by an active form of cytosolic T3-binding protein in vitro.

Epoxide hydrolysis in the cytosol of rat liver, kidney, and testis: Measurement in the presence of glutathione and the effect of dietary clofibrate

The hydrolysis of trans- and cis-stilbene oxide and benzo[ a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione-conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[ a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes.

Evidence for the secretion of an antimineralocorticoid in congenital adrenal hyperplasia.

Plasma extracts from patients with congenital adrenal hyperplasia were found to contain substances that competed with aldosterone for mineralocorticoid receptor-binding sites in a rat kidney cytosol system. In normal subjects and patients with other disorders, the mineralocorticoid receptor-binding activity in such extracts could be entirely accounted for by the sum of the contributions of the steroids known to bind to the mineralocorticoid receptor. The secretion of these binding substances in patients with the C-21 hydroxylation defect was ACTH dependent. While these substances could be either mineralocorticoid agonists or antagonists, the latter is more likely. Production of mineralocorticoid antagonists would account for the compensatory hyperaldosteronism that occurs in the simple virilizing form, in which there is minimal impairment of aldosterone secretory reserve, and for the tendency to Addisonian crisis in patients with the salt-losing form, who have a more severe defect in aldosterone biosynthesis.

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Mineralcorticoid receptor from rat kidney. Interaction with heparin and purification to a CBG-free stage

Purification of the mineralcorticoid receptor is a particularly challenging problem. This receptor is present in target tissues at concentrations lower and is less stable than any other steroid receptor. Addition of molybdate ions (20 mM) to rat kidney cytosol enhances stability of mineralcorticoidspecific binding sites: the inactivation rate at 0°C decreases from 7.2 to 1.7% per hour in the absence of aldosterone, and from 1.8 to 0.3% per hour in the presence of hormone. Rates of inactivation in the presence of molybdate are thus compatible with purification procedures. Also, the corticosteroid-binding globulin (CBG) is an important contaminating component of kidney eytosol because it cannot be specifically blocked preliminarily to affinity chromatography. We show that when kidney cytosol is incubated with heparin covalently linked to Sepharose (Sepharose-heparin), after 30 min at 0°C more than 80% of the mineralcorticoid-specific binding sites interact strongly with Sepharose-heparin while CBG is not bound at all. The mineralcorticoid-specific binding sites can be recovered from Sepharose-heparin by washing with heparin (2 mg/ml; recovery up to 90%), KCl (0.3 M; recovery up to 90%); and, less efficiently, with total liver RNA (2 mg/ml; recovery up to 55%) and dextran sulfate (2 mg/ml; recovery up to 40%); little or no recovery is achieved with chondroitin sulfate, sonicated DNA, pyridoxal-5-phosphate, dextran, d-glucosamine and d-glucuronic acid. With demonstration that also the mineralcorticoid receptor binds to heparin, this property has become a general hallmark of steroid receptors. If the “heparin” binding site of steroid receptors is of physiological significance it remains to be established. By application of the newly found property of the mineralcorticoid receptor, an overall 10-fold purified, CBG-free preparation of this receptor can be obtained from kidney cytosol with a single chromatography on Sepharose-heparin.

Isoenzymes of glutathione transferase in rat kidney cytosol.

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a 'new' form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues

Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate K., Nakagomi K. and Nakazawa Y. (1984) J. Biol. Chem. 259, 14706–14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3–0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.

Purification and characterization of a cytosolic protein enhancing GSH-dependent microsomal iodothyronine 5'-monodeiodination.

A protein has been purified from rat liver cytosol which promoted GSH-responsive iodothyronine 5'-deiodinase activities in rat kidney microsomes. The factor behaved as a basic protein with an Mr of 11,000. It was active as a GSH-disulfide transhydrogenase with beta-hydroxyethyl disulfide as an acceptor and was also active in stimulating calf thymus ribonucleotide reductase with one-third the potency of native calf thymus glutaredoxin. Another basic protein, which degraded iodothyronines oxidatively, was also identified in the cytosolic preparations; this co-purified with soluble protein factor in the earlier purification stages and was partially separated from this factor by CM-cellulose chromatography. The glutaredoxin-like protein present in rat liver and kidney cytosol could provide a physiologic regulatory mechanism for GSH-dependent 5'-monodeiodination of iodothyronines.