Hepatic Nuclear Envelopes Research Articles

Phospholipase A 2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A 2 (PLA 2) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA 2 (10 −5 ∼ 10 −2/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [ 3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA 2, regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA 2-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA 2-treated group. Messenger RNA export rate in PLA 2 (10 −3 unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA 2 plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.

Effects of ascorbic acid on hepatic nuclear binding of L-tryptophan

This study investigated the in vitro effects of ascorbic acid on nuclear L-tryptophan receptor binding of rat liver. Ascorbic acid at 10 −6M to 10 −4M inhibited the in vitro 3H-tryptophan binding to hepatic nuclei and nuclear envelope receptors. Addition of dithiothreitol, a protective agent for sulfhydryl groups, did not affect the inhibitory binding. Addition of L-leucine did reduce the inhibitory binding. Addition of NaCl (0.125 × 10 −4M) did not influence the inhibitory binding effect of ascorbic acid but decreased the inhibitory effect of the NaCl on nuclear tryptophan binding. In vivo administration of ascorbic acid before or along with L-tryptophan decreased the L-tryptophan-induced stimulation of hepatic protein synthesis.

Effects of altered tonicity by sodium chloride on L-tryptophan binding to hepatic nuclei.

This study was concerned with the effects of NaCl administered in vivo or added in vitro to isolated nuclei on [(3)H]tryptophan binding to rat hepatic nuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivo to rats caused at 10 min a marked decrease in in vitro binding (total and specific) of [(3)H]tryptophan to hepatic nuclei. In vitro incubation of isolated hepatic nuclei, but not of isolated nuclear envelopes, with added NaCl (particularly at 0.125 x 10(-4) M and 0.25 x 10(-4) M) revealed significant inhibition of [(3)H]tryptophan binding. However, isolated hepatic nuclear envelopes prepared after in vitro incubation of isolated nuclei with added NaCl did show inhibition of [(3)H]tryptophan binding (total and specific) compared with controls. Other salts (KCl, MgCl(2), NaHCO(3), NaC(2)H(3)O(2), NaF, or Na(2)SO(4)), at similar concentrations to that of NaCl except for MgCl(2), when added to isolated nuclei did not appreciably inhibit nuclear tryptophan binding. Kinetic studies of in vitro nuclear [(3)H]tryptophan binding in the presence of 0.125 x 10(-4) M NaCl revealed that binding decreased at 0.5 h and continued to 2 h compared with nuclear [(3)H]tryptophan binding with controls (without NaCl addition). The results obtained in vivo in rats and those obtained in vitro with isolated hepatic nuclei revealed NaCl-induced inhibitory effects on [(3)H]tryptophan binding to hepatic nuclei. Although the inhibitory effects were similar under the two different experimental conditions, the mechanism for each may be different in that the NaCl concentration in hepatic cells after administration of NaCl in vivo was appreciably higher than the low levels added in vitro to the isolated hepatic nuclei.

Effect of cycloheximide on tryptophan binding to rat hepatic nuclei.

This study evaluated whether cycloheximide, an inhibitor of protein synthesis, would affect the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. Also, the administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we determined that the addition of cycloheximide in vitro inhibited 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. Heat-treated cycloheximide failed to have this inhibitory binding effect. In vivo treatment of rats with cycloheximide diminished in vitro 3H-tryptophan binding to hepatic nuclei of treated rats compared to controls. Puromycin, another inhibitor of hepatic protein synthesis, when added in vitro did not affect 3H-tryptophan binding to hepatic nuclei but did diminish in vitro binding after in vivo treatment. Thus, cycloheximide added in vitro diminished 3H-tryptophan binding to hepatic nuclei probably by its structural effect on the receptor while cycloheximide administered in vivo may also act in part by inhibiting protein synthesis.

Hormonal influences on tryptophan binding to rat hepatic nuclei

This study evaluated whether selected hormones, 3,5,3'-triiodothyronine (T3), hydrocortisone (HC), or insulin, would influence the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. The first two hormones have nuclear receptors that belong to the same superfamily, while insulin belongs to a different unrelated superfamily of receptors. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear proteins was saturable, stereospecific, and of high affinity. Also, previous studies showed that administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we investigated whether each hormone alone or together with unlabeled tryptophan would influence tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro L-5-(3)H-tryptophan binding. Our results indicate that T3 10(-14) to 1(-10) mol/L appreciably inhibited in vitro 3H-tryptophan binding to hepatic nuclei and T3 10(-16) to 10(-4) mol/L appreciably ameliorated the inhibitory effect of unlabeled tryptophan (10(-4) mol/L) on in vitro 3H-tryptophan binding. In vivo administration (1 hour) of tryptophan alone stimulated hepatic protein synthesis, but addition of T3 negated such stimulation. Addition of HC 10(-12) to 10(-4) mol/L had no effect and addition of insulin 10(-16) to 10(-4) mol/L had only a small inhibitory effect on in vitro 3H-tryptophan binding to rat hepatic nuclei, but each (10(-12) to 10(-4) mol/L), when added to unlabeled tryptophan (10(-4) mol/L), diminished the inhibitory binding effect of unlabeled tryptophan alone. Our study indicates that T3 competes with tryptophan for hepatic nuclear tryptophan binding, and it also appears to negate tryptophan's stimulatory effect on hepatic protein synthesis.

Hormonal influences on tryptophan binding to rat hepatic nuclei.

We evaluated whether selected hormones, 3,5,3'-triiodothyronine (T3), hydrocortisone (HC) or insulin, would influence the binding (saturable, stereospecific, and of high affinity) of L-tryptophan to rat hepatic nuclei or nuclear envelopes. T3 (10(-14) to 10(-10) M) appreciably inhibited in vitro L-(5-3H) tryptophan binding to hepatic nuclei and T3 (10(-16) to 10(-4) M) appreciably ameliorated the inhibitory effect of unlabeled tryptophan (10(-4) M) on such binding. In vivo tryptophan administration (1 h) stimulated hepatic protein synthesis but the addition of T3 negated such stimulation. HC (10(-12) to 10(-4) M) did not affect and insulin (10(-16) to 10(-4) M) had only a small inhibitory effect on 3H-tryptophan binding to hepatic nuclei, but each (10(-12) to 10(-4) M) when added to unlabeled tryptophan (10(-4) M) diminished the inhibitory binding effect of unlabeled tryptophan alone. Thus, T3 competes with tryptophan for hepatic nuclear tryptophan binding and also negates tryptophan's stimulatory effect on hepatic protein synthesis.

Tryptophan binding to nuclei of rat liver and hepatoma

This study was conducted to determine whether L-tryptophan binding to nuclei of rat transplantable hepatomas (5123 and 19) differed from that in host liver or normal liver of rats. Binding of L-tryptophan to rat hepatic nuclear proteins has been demonstrated to be saturable, stereospecific, and of high affinity and the nuclear envelope binding protein has been purified and characterized. Using an in vitro 3H-tryptophan binding assay, the total and specific L-tryptophan binding was appreciably less in nuclei of hepatoma than in nuclei of host liver or normal liver. On Scatchard analyses, the K D values were similar for nuclei of hepatoma and of host liver but the B max values were less in hepatoma than in host liver. Free L-tryptophan levels and nuclear poly(A)polymerase activity levels in hepatoma were higher than those of host liver. Using compounds (L-tryptophan implicated in the eosinophilia-myalgia syndrome, D,L-β-(1-naphthyl)alanine, chlordiazepoxide, and 3-methylindole) that had earlier been found to diminish in vitro 3H-tryptophan binding to rat hepatic nuclei, similar inhibitory effects were observed with nuclei of hepatoma or with nuclei of host liver. Using polyclonal antibodies raised against the L-tryptophan binding protein of rat hepatic nuclear envelopes, immunoblot assay was performed on nuclei of hepatoma and of normal liver. The receptor protein (67kD) was present in both preparations.

Comparative studies on tryptophan binding to hepatic nuclear envelopes in Sprague-Dawley and Lewis rats

Since Lewis rats are susceptible to many inflammatory diseases and have been used in an experimental model of the eosinophilia-myalgia syndrome, we investigated whether Lewis rats would respond to L-tryptophan as have Sprague-Dawley rats reported earlier. In this comparative study using females of both strains, we observed a decrease in the affinity of in vitro L-tryptophan binding to hepatic nuclei and nuclear envelopes of Lewis rats compared with Sprague-Dawley rats. However, in vivo stimulatory effects of administering L-tryptophan on hepatic polyribosomal aggregation, protein synthesis, and nuclear RNA release were similar in both strains. In vitro [3H]tryptophan binding to hepatic nuclear envelopes, using L-tryptophan implicated in cases of the eosinophilia-myalgia syndrome, revealed less specific binding than when using nonimplicated L-tryptophan in both strains. The possible significance of the quantitative difference in the binding affinity of L-tryptophan to hepatic nuclei of Lewis rats compared with those of Sprague-Dawley rats is as yet undetermined.

Indolic Compounds Affect Tryptophan Binding to Rat Hepatic Nuclei

This study evaluates the effects of indolic or indole-related compounds on binding of L-tryptophan (saturable, stereospecific and of high affinity) to rat hepatic nuclei or nuclear envelopes. Addition of any one of many indolic or indole-related compounds, and particularly of 3-methylindole, does not inhibit in vitro binding of [3H]tryptophan to hepatic nuclear envelopes. However, when 3-methylindole (10(-10) to 10(-4) mol/L) is added in combination with unlabeled L-tryptophan (10(-4) mol/L), it diminishes the inhibitory effect of unlabeled L-tryptophan alone. Scatchard analysis of the binding affinities of in vitro [3H]tryptophan binding to hepatic nuclear envelopes using L-tryptophan in the absence or presence of 3-methylindole reveals similar dissociation constants (KD) under the two conditions, but the binding concentrations (Bmax) were greater in the combined group compared with that in the L-tryptophan alone group. Addition of 3-methylindole to liver before homogenization decreases specific [3H]tryptophan binding to nuclei compared with controls (without addition). L-Tryptophan tube-fed to rats with or without 3-methylindole administration increases in vitro hepatic protein synthesis compared with that of saline tube-fed controls. 3-Methylindole itself does not affect protein synthesis. Our report describes the effects of 3-methylindole on specific tryptophan binding to hepatic nuclear envelope receptor and discusses the possible implications thereof.

Studies with 1,1′-Ethylidenebis(tryptophan), a Contaminant Associated with L-Tryptophan Implicated in the Eosinophilia-Myalgia Syndrome

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [ 3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [ 3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1′-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [H-3]-tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [ 3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [ 14C]leucine incorporation into proteins. [ 14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented.

Effect of 3-phenylamino- l-alanine on tryptophan binding to rat hepatic nuclear envelopes

We have determined that the addition of 3-phenylamino- l-alanine (PAA), a recently reported contaminant in l-tryptophan implicated in the eosinophilia-myalgia syndrome, affects tryptophan binding by utilizing an in vitro measurement of 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. PAA (10 −10 to 10 −4M) diminishes the inhibitory effect of binding due to excess unlabeled l-tryptophan (10 −4M). PAA alone has no inhibitory effect on binding. The effect of PAA on in vitro tryptophan binding is in contrast to that of another contaminant, 1,1′-ethylidenebis(tryptophan), which together with excess unlabeled l-tryptophan does not appreciably affect the binding. In vitro addition of PAA and l-tryptophan to nuclei of rat brain or of cultured murine macrophages does not affect [ 3H]tryptophan binding in comparison to l-tryptophan alone as if the case with hepatic nuclear envelopes. Adding PAA to an in vitro protein synthesis system and measuring [ 3H]tryptophan or [ 3H]alanine incorporation into acid-precipitable proteins reveals that it competes similarly, but somewhat less, than does equimolar concentrations of unlabeled l-tryptophan or l-alanine, respectively. This suggests that PAA or a breakdown compound becomes incorporated into proteins. Speculation as to how PAA may affect tissues in experimental animals is presented.

Effect of metyrapone on tryptophan binding to rat hepatic nuclei

This study evaluated whether metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), an inhibitor of endogenous adrenal corticosteroid synthesis via inhibition of cytochrome P-450-mediated steroid hydroxylation, would influence the binding of l-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous publications have indicated that binding of l-tryptophan to hepatic nuclear envelope proteins was saturable, stereospecific, and of high affinity. In this study, we investigated whether metyrapone would influence l-tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro l-(5- 3H)tryptophan binding. Our results indicate that the addition of metyrapone in vitro has little influence on l-(5- 3H)tryptophan binding to hepatic nuclei or nuclear envelopes. On the other hand, when metyrapone ( 1 mg 100 g body weight ) is tube-fed 30 minutes before killing, the isolated hepatic nuclei show decreased specific l-tryptophan binding (total binding minus nonspecific binding [using 2,000-fold excess of unlabeled l-tryptophan]) compared with controls. Also, addition of metyrapone in vitro to rat liver before homogenization and preparation of nuclei caused the nuclei to show decreased specific tryptophan binding compared with controls. Under these in vitro conditions, SKF 525A, another inhibitor of hydroxylation, showed inhibitory effects similar to those of metyrapone. Thus, metyrapone can interfere with rat liver nuclear envelope receptor binding to l-tryptophan, and possibly acts via its effects on hydroxylation. At high doses, metyrapone ( 20 mg 100 g body weight ) appears to inhibit tryptophan-induced stimulation of hepatic protein synthesis.

Competitive studies relating to tryptophan binding to rat hepatic nuclear envelopes as a sensitive assay for unknown compounds

Our laboratory has reported that L-tryptophan binds to a rat liver nuclear envelope protein and this binding is saturable, stereospecific and of high affinity. Utilizing an in vitro [ 3H]tryptophan binding assay to hepatic nuclear envelopes, we have determined the effects of using excess unlabeled L-tryptophan from a number of different suppliers. This study reports that, based on our in vitro binding assay, some significant differences were observed when implicated L-tryptophan in cases of the eosinophilia-myalgia syndrome obtained from a Japanese manufacturer, Showa Denko, was assayed, in contrast to non-implicated L-tryptophan from other suppliers. An isolated impurity of Showa Denko L-tryptophan, 1,1′-ethylidenebis(tryptophan) alone or together with non-implicated L-tryptophan or its breakdown product, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, did not appreciably affect the in vitro [ 3H]tryptophan binding to hepatic nuclear envelopes as did the Showa Denko L-tryptophan. Our data, derived with our in vitro binding assay system, suggests that implicated L-tryptophan from Showa Denko contains a compound/s (unknown at present) other than 1,1′-ethylidenebis(tryptophan), which alters in vitro [ 3H]tryptophan binding. The significance of the impurity/ies involved remains to be determined.

Association of poly(A)polymerase with tryptophan receptor in rat hepatic nuclei

This study conducted experiments with two rat liver nuclear envelope proteins, a tryptophan receptor glycoprotein and poly(A)polymerase, and compared the structural similarities by using affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and antibody specificity. Earlier, a rat hepatic nuclear envelope glycoprotein that is present in limited quantity and binds tryptophan with high affinity was identified and characterized. Also, analysis of eluted nuclear membrane proteins bound to either tryptophan-agarose or concanavalin A-agarose columns by SDS-PAGE previously revealed a major protein of similar molecular weight after staining with Coomassie blue. Polyclonal antibodies were raised in rabbits by injecting the excised gel protein band. The second protein, an enzyme, poly(A)polymerase, was previously purified from rat liver nuclei by sequential chromatography on DEAE-Sephadex, QAE-Sephadex, phosphocellulose, hydroxyapatite, and DNA-cellulose columns. Analysis of eluate from the final column by SDS-PAGE and Coomassie blue staining revealed a single band. Antibodies to the purified enzyme were raised in chickens. In the present study, using anti-tryptophan receptor antibodies and anti-poly(A)polymerase antibodies, we observed that each recognized the same protein by immunoblot analysis. The anti-tryptophan receptor antibodies did not recognize the catalytic site of poly(A)polymerase but did reduce the enzymatic activity when the antigen-antibody complex was sedimented using Pansorbin ( Staphyloccus aureus cells wearing a coat of protein A). However, antipoly(A)polymerase antibodies inhibited binding of 3H-tryptophan to the receptor site. Furthermore, tryptophan could elute poly(A)polymerase from both tryptophan agarose and poly(A)sepharose columns. Analysis of eluates from these columns by SDS-PAGE followed by silver staining of the gel revealed the presence of a major band with an apparent molecular weight of about 65,000–67,000. Our present findings suggest that the tryptophan receptor and poly(A)polymerase share structural homology.

Inhibitory effect of demoxepam on tryptophan binding to rat hepatic nuclei.

Since some patients with eosinophilia-myalgia syndrome ingested tryptophan along with benzodiazepines, we investigated whether demoxepam, the N-desalkylated compound of chlordiazepoxide, would influence the binding of tryptophan to hepatic nuclei. L-Tryptophan has been shown to bind (saturable, stereospecific, and of high affinity) to rat hepatic nuclei and nuclear envelopes. We report that demoxepam has an inhibitory effect on in vitro [3H]tryptophan binding to rat hepatic nuclei and has an apparent KD approximately 22 microM.

Studies with Compounds that Compete with Tryptophan Binding to Rat Hepatic Nuclei

Tryptophan has been demonstrated to affect hepatic RNA and protein metabolism. Binding of tryptophan to nuclear envelope proteins has been demonstrated to be saturable, stereospecific, and of high affinity. The hepatic nuclear envelope tryptophan binding protein (glycoprotein) has been purified to apparent homogeneity using either concanavalin A-agarose or tryptophan-agarose. The receptor has an Mr of ∼34,000, which is the same as that observed when [3H]tryptophan has been crosslinked to nuclear proteins. In this study, we investigated whether analogs, metabolites or related compounds of tryptophan as well as other amino acids may bind to rat hepatic nuclei using in vitro [3H]tryptophan binding assays. Our results indicate that compounds that compete with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes contain the α-aminopropionic acid structure. Such compounds were 5-fluoro tryptophan, 7-aza tryptophan, 5-hydroxy tryptophan, alanine, phenylalanine, tyrosine, cysteine and cystine. It was of interest that, whereas tryptophan-methyl ester and tryptophan-ethyl ester competed, α-methyl tryptophan, N-formyl tryptophan, N-acetyl tryptophan, and N-methyl tryptophan did not compete with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes. Nonetheless, only the in vivo administration of L-tryptophan was able to stimulate nucleocytoplasmic efflux of hepatic RNA and protein synthesis.

Effect of benzodiazepines on tryptophan binding to rat hepatic nuclei.

This study evaluates whether or not some of the benzodiazepines would influence the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous publications have indicated that binding of L-tryptophan to hepatic nuclear envelope proteins was saturable, stereospecific, and of high affinity. In this study, we investigated whether some of the benzodiazepines would influence L-tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitro L-(5-3H) tryptophan binding. Our results indicate that the addition of chlordiazepoxide, diazepam, prazepam, flurazepam, nordazepam, N-desalkylflurazepam, temazepam, oxazepam, lorazepam, or 4-chlorodiazepam has little influence on the L-(5-3H) tryptophan binding to hepatic nuclei in vitro. However, the addition of demoxepam, the N-desalkylated compound of chlordiazepoxide, caused marked competition with 3H-tryptophan binding to hepatic nuclei in vitro. When chlordiazepoxide (1 mg/100 g body weight) is administered intraperitoneally 20 min before killing, the isolated hepatic nuclei reveal decreased specific L-tryptophan binding compared to controls. Also, rats pretreated with chlordiazepoxide intraperitoneally before tube-feeding L-tryptophan revealed diminished tryptophan-induced hepatic nuclear RNA efflux and protein synthesis. Our results suggest that chlordiazepoxide, possibly by itself or through a metabolite, can act to affect hepatic nuclear binding of L-tryptophan and to inhibit the stimulatory effect of L-tryptophan on hepatic protein synthesis.

Prevention of 2-acetylaminofluorene-induced loss of nuclear envelope cytochrome P450 by the simultaneous administration of 3-methylcholanthrene

Rats fed a basal diet containing 0.05% (w/w) 2-acetylaminofluorene (AAF) for 3 weeks showed a 50% loss of hepatic nuclear envelope cytochrome P450, whereas microsomal P450 remained at control levels. A similar dietary treatment with 0.004% (w/w) 3-methylcholanthrene (MC) caused moderate losses (20–25%) of cytochrome P450 in both nuclear envelopes and microsomes. Administration of the basal diet supplemented with a mixture of AAF (0.05%) plus MC (0.004%) resulted in a preservation of control levels of nuclear envelope cytochrome P450 and a 30% elevation of microsomal P450. Immunoblot analysis revealed that AAF alone, or in concert with MC, induced comparable levels of the P450d form. Induction of cytochrome P450c by dietary MC was detected only when MC was fed together with AAF. As previously found for butylated hydroxytoluene (BHT), the protective effect of dietary MC against hepatocarcinogenesis in AAF-fed rats correlated with a preservation of nuclear envelope cytochrome P450 content and with the induction of cytochrome P450c.

Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver: isolation, characterization, and immunohistochemical localization.

A protein with poly(A) polymerase activity has been identified and isolated from hepatic nuclear envelopes of rats to near homogeneity. The ability of the enzyme to bind to concanavalin A-agarose and to be eluted from the column with methyl alpha-D-mannopyranoside (0.2 M) as well as the inhibitory effects of alpha-mannosidase suggested that it was a glycoprotein. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP, and an oligonucleotide primer. The enzyme activity with Mn2+ was about 20-fold higher than that with Mg2+. Several known inhibitors adversely affected poly(A) polymerase activity. The enzyme has a molecular weight of 64,000 when analyzed by polyacrylamide gel electrophoresis under denaturing conditions and has a sedimentation coefficient of 4.5 S. Immunohistochemical studies using polyclonal antibodies raised against the purified enzyme revealed that the antigen was localized in the nuclear membranes.

The influence of tryptophan on hepatic polyribosomes and protein synthesis in fasted mice

This study evaluated whether selected hormones, 3,5,3′-triiodothyronine (T3), hydrocortisone (HC), or insulin, would influence the binding ofl-tryptophan to rat hepatic nuclei or nuclear envelopes. The first two hormones have nuclear receptors that belong to the same superfamily, while insulin belongs to a different unrelated superfamily of receptors. Previous reports have indicated that the binding ofl-tryptophan to hepatic nuclear proteins was saturable, stereospecific, and of high affinity. Also, previous studies showed that administration ofl-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we investigated whether each hormone alone or together with unlabeled tryptophan would influence tryptophan binding to rat hepatic nuclei or nuclear envelopes as assayed by in vitrol-5-3H-tryptophan binding. Our results indicate that T3 10−14 to 10−10 mol/L appreciably inhibited in vitro3H-tryptophan binding to hepatic nuclei and T3 10−16 to 10−4 mol/L appreciably ameliorated the inhibitory effect of unlabeled tryptophan (10−4 mol/L) on in vitro3H-tryptophan binding. In vivo administration (1 hour) of tryptophan alone stimulated hepatic protein synthesis, but addition of T3 negated such stimulation. Addition of HC 10−12 to 10−4 mol/L had no effect and addition of insulin 10−16 to 10−4 mol/L had only a small inhibitory effect on in vitro3H-tryptophan binding to rat hepatic nuclei, but each (10−12 to 10−4 mol/L), when added to unlabeled tryptophan (10−4 mol/L), diminished the inhibitory binding effect of unlabeled tryptophan alone. Our study indicates that T3 competes with tryptophan for hepatic nuclear tryptophan binding, and it also appears to negate tryptophan's stimulatory effect on hepatic protein synthesis.