Hepatic iodothyronine 5''-deiodinase. The role of selenium.

Selenium (Se) deficiency decreased by 8-fold the activity of type 1 iodothyronine 5'-deiodinase (ID-I) in hepatic microsomal fractions from rats. Solubilized hepatic microsomes from rats injected with 75Se-labelled Na2SeO3 4 days before killing were found by chromatography on agarose gels to contain a 75Se-containing fraction with ID-I activity. PAGE of this fraction under reducing conditions, followed by autoradiography, revealed a single 75Se-containing protein (Mr 27,400 +/- 300). This protein could also be labelled with 125I-bromoacetyl reverse tri-iodothyronine, an affinity label for ID-I. The results suggest that hepatic ID-I is a selenoprotein or has an Se-containing subunit essential for activity.     

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5''-deiodinase.

The properties and kinetic characteristics of a non-GSH NADPH-dependent cofactor system activating rat hepatic and renal 5'-deiodinase (5'-DI), which we have previously demonstrated with partially purified cytosol Fractions A and B [Sawada, Hummel & Walfish (1986) Biochem. J. 234, 391-398], were examined further. Although microsomal fractions prepared from either rat liver or kidneys could be activated by crude cytosol Fractions A and B from those tissues as well as from rat brain and heart, a homologous hepatic or renal system was the most potent in producing 5'-deiodination of reverse tri-iodothyronine (rT3). At nanomolar concentrations both rT3 and thyroxine (T4) were deiodinated but with a much greater substrate preference for rT3 than for T4. However, at micromolar concentrations of these substrates no activation of 5'-DI could be detected. In this deiodinative system, T4 and tri-iodothyronine (T3) competitively inhibited 5'-deiodination of rT3. Dicoumarol, iopanoate, arsenite and diamide were also inhibitory to the activation of hepatic or renal 5'-deiodination by this cofactor system. Purification of cofactor components in hepatic crude cytosolic Fractions A and B to near homogeneity, as assessed by their enzymic and physical properties, indicated that these co-purified with and were therefore identical with thioredoxin reductase and thioredoxin respectively, and accounted almost entirely for the observed activation of rT3 5'-DI. When highly purified liver cytosolic thioredoxin reductase and thioredoxin were utilized to determine the kinetic characteristics of the reaction, evidence for a sequential mechanism operative at nanomolar but not micromolar concentrations of rT3 and T4 was obtained. The Km for rT3 was 1.4 nM. Inhibition by 6-n-propyl-2-thiouracil (Ki 6.7 microM) was competitive with respect to thioredoxin and non-competitive with respect to rT3, whereas inhibition by T4 (Ki 1.3 microM) was competitive. Since rT3 is a potent inhibitor of T4 5'-deiodination, this thioredoxin system activating deiodination of rT3 may play an important role in regulating the rate of intracellular production of T3 from T4.     

Structural requirements of thiol compounds in the inhibition of human liver iodothyronine 5''-deiodinase.

2-Thiouracil and a number of its alkyl derivatives are known to inhibit the enzymic 5'-deodination of thyroxine to 3,5,3'-tri-iodothyronine. The structural requirements for inhibition of iodothyronine 5'-deiodinase were investigated by using a washed postmitochondrial particulate fraction of human liver. A series of sulphur-containing derivatives of pyrimidine, pyridine, imidazole, benzene and urea, capable of existing in a thiol form, were incubated at several concentrations with the enzyme preparation in the presence of thyroxine and dithioerythritol (cofactor). The degree of inhibition by the respective compounds of the production of 3,5,3'-tri-iodothyronine was studied in relation to their structural features. The major observations were: (i) a free thiol group is essential; (ii) compounds that do not possess a polar hydrogen atom spatially configured so that it is proximal to the thiol group are poor inhibitors; (iii) aromatic characteristics in the presence of requirements (i) and (ii) lead to the expression of potent inhibitory properties; (iv) modification of potent inhibitors by the introduction of hydrophilic substituents reduces the inhibitory potency.     

Binding of diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions in vitro and in mouse foetal cells in culture.

Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.     

Biochemical and cytochemical localization of inosine-5''-diphosphatase in rat liver microsomal fractions.

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.     

NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5''-deiodinase. Demonstration by alkylation with iodoacetamide.

We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B.     

Palmitate activation and esterification in microsomal fractions of rat liver.

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.     

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.     

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

Cyclic metabolic pathway of a butylated hydroxytoluene by rat liver microsomal fractions

A cyclic metabolic pathway was obtained when 3,5-di-t-butyl-4-hydroxytoluene (BHT) was incubated with a rat liver microsomal preparation. The pathway is as follows: BHT --> 4-hydroperoxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-OOH) --> 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-3(0)OH) --> BHT. This metabolic pathway suggests that antioxidants such as BHT owe their high efficacy, at least in part, to their metabolic regeneration.     

The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

Binding of inositol trisphosphate by a liver microsomal fraction.

To test the hypothesis that inositol trisphosphate (InsP3) mediates adaptation and excitation in invertebrate photoreceptors, we measured its formation on a rapid time scale in squid retinas. For squid, excitation and adaption occurs within 0.1 and 1-2 s respectively. We could detect an elevation in InsP3 within 200 ms of a bright flash. This increase is about 240% over dark basal levels and is maintained for at least 2 min after a flash. The increase probably occurs in the photoreceptors, which are the only neurons in squid retinas. Analysis by h.p.l.c. indicates that the light-regulated isomer is Ins(1,4,5)P3, which is formed by the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2).     

Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

E.s.r. spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect peroxyl, alkoxyl and carbon-centred radicals produced by reaction of t-butyl hydroperoxide (tBuOOH) with rat liver microsomal fraction. The similarity of the hyperfine coupling constants of the peroxyl and alkoxyl radical adducts to those obtained previously with isolated enzymes suggests that these species are the tBuOO. and tBuO. adducts. The effects of metal-ion chelators, heat denaturation, enzyme inhibitors and reducing equivalents demonstrate that these species arise from reaction of tBuOOH with a haem enzyme such as cytochrome P-450 or cytochrome b5. In the absence of NADPH or NADH the previously undetected peroxyl radical adduct is the major species observed. In the presence of these reducing equivalents the alkoxyl and carbon-centred radical adducts predominate, which is in accord with product studies on similar systems. These results demonstrate that both reductive and oxidative decomposition of tBuOOH can occur in rat liver microsomal fraction with the reductive pathway favoured in the presence of NADH or NADPH.     

Metabolism of DHEA by cytochromes P450 in rat and human liver microsomal fractions

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.     

Synthesis of rat liver microsomal cytochrome b5 by free ribosomes

Free and membrane-bound polyribosomes were separated from liver homogenates and characterized by electron microscopy. Using the wheat germ cell-free translation system, total translation products of poly A+RNA extracted from free polyribosomes (poly A+RNAf) showed some correlation to total liver cytosol proteins. In contrast, translation products of poly A+RNA from membrane-bound polyribosomes (poly A+RNAmb) showed some similarity to rat serum. Antibody to purified rat serum albumin immunoprecipitated from only the translation products of poly A+RNAmb a single polypeptide of mol wt 68,000. i.e., 3,000 greater than secreted serum albumin. In contrast, antibody to detergent-extracted cytochrome b5 immunoprecipitated from only the translation products of poly A+RNAf a single polypeptide of mol wt 17,500, identical to that of microsomal cytochrome b5. A consideration of the known properties of cytochrome b5 is consistent with an exclusive site of synthesis on free ribosomes.