An acute phase response factor/NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma.

The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect, we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and evaluated the IL-6 inducibility of these sequences by transient expression in an IL-6-dependent plasmacytoma cell line. Although a 6.5-kilobase fragment of upstream flanking sequence did not increase the IL-6 inducibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down-stream flanking sequence that both up-regulates the promoter and confers inducibility by IL-6. Point mutation of an acute phase response factor (APRF) site within this region significantly reduced up-regulation of the promoter in cells grown continuously in IL-6, as well as inducibility upon restimulation of cells with IL-6 after withdrawal from the growth factor. Point mutation of an NF-kappa B site sharing five nucleotides with the APRF site reduced up-regulation of the promoter but not inducibility by IL-6, whereas mutation of two other NF-kappa B sites in the 222-base pair fragment had no effect on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and constitutive binding of NF-kappa B p65 to the APRF/NF-kappa B site.

Production of delta-aminolevulinic acid: characterization of murine liver 4,5-dioxovaleric acid: L-alanine aminotransferase.

1. We report on the kinetic properties of murine liver 4,5-dioxovaleric acid:L-alanine aminotransferase (DOVA transaminase). 2. The transamination of 4,5-dioxovaleric acid (DOVA) led to the production of delta-aminolevulinic acid. 3. L-Alanine was the preferred amino group donor among the common 20 amino acids. 4. The optimum pH of the reaction was 7-8. 5. A Km of 220 microM for DOVA and a Km of 970 microM for L-alanine were obtained. 6. The reaction was inhibited by each of the following: glyoxylate, beta-chloroalanine, methylglyoxal, delta-aminolevulinate, pyruvate, heme, and gabaculine. 7. None of several xenobiotic inducers of microsomal mixed function oxidases tested had a significant effect on DOVA transaminase activity in studies performed with murine primary hepatocyte cultures.

Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase.

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.

Mitochondrial biogenesis: do liver mitochondria contain glycoproteins and glycosyltransferases?

1. Subcellular fractions isolated from livers of 19-day-old chicken embryos were analyzed in order to assess whether liver mitochondria contained glycosylated proteins or had mannosyl- or sialyl-transferases that could transfer sugars to mitochondrial macromolecules. 2. Proteins in liver mitochondrial membranes and matrix fractions were screened for their affinities for concanavalin A (Con A). 3. After separation by gel electrophoresis under denaturing conditions, a significant number of the proteins bound [125I]Con A, and the binding of the lectin was substantially inhibited by alpha-methyl-D-mannoside. 4. In addition, radio-iodinated matrix proteins were screened for lectin-binding properties by chromatography on Con A covalently linked to agarose. 5. A number of proteins, representing 14% of those loaded onto the column, became tightly bound to the agarose-linked lectin, and the molecular weights of several of those proteins are reported. 6. Mannosyltransferase activities were measured in fractions highly enriched for mitochondria. 7. In the reactions, mannose was transferred from guanosine diphosphomannose to materials insoluble in 0.3% trichloroacetic acid or in chloroform:methanol (2:1). 8. The fractions also catalyzed the transfer of mannose to materials extractable in chloroform:methanol and which migrated with the Rf of dolichol phosphate on Silica Gel H. 9. Dolichol phosphate stimulated the transfer of mannose to those materials extractable in the organic solvents. 10. Marker enzyme analyses indicated that the mannosyl transferase activity in the mitochondrial fraction could not be accounted for entirely by contaminating microsomal membranes. 11. Although sialyltransferase activity was detected also in the mitochondrial fractions, the levels of the activity and the kinetics of the reactions indicated that Golgi membranes were most likely the sources of the enzyme.

Induction of cytochrome P-450 RNA by porphyrogenic agents: on the nature of the coordinate induction with delta-aminolevulinate synthase in tissues of the chicken embryo.

1. We determined by cDNA-RNA solution hybridization analyses that in ovo administration of allylisopropylacetamide in combination with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate increased the concentrations of cytochrome P-450 RNA in liver, kidney, and intestine of 18-day-old chicken embryos. 2. Similarly, the administration of testosterone to embryos caused elevations in the cytochrome P-450 RNA levels in liver and kidney. 3. The increases in cytochrome P-450 RNA concentrations occurred only in those tissues where elevations in delta-aminolevulinate (ALA) synthase activity and mRNA content were measured (liver, kidney and intestine) but not in tissues where the activity and RNA levels of ALA synthase did not change (heart, brain, lung). 4. The increases in the concentrations of the cytochrome P-450 RNA were not affected by loading embryos with ALA and FeCl3 at the time of administration of the inducers.

Regulation of production of delta-aminolaevulinate synthase in tissues of chick embryos. Effects of porphyrogenic agents and of haem precursors.

Studies conducted by several groups have established that porphyrogenic agents which caused elevations in chick-embryo liver delta-aminolaevulinate (ALA) synthase activity also increased the concentrations of the enzyme's RNA, and that haemin inhibited these elevations. We have determined in this study, using immune-blot analyses, that administration in ovo of allylisopropylacetamide (AIA) in combination with diethyl 1,4-dihydro-2,4,6-trimethyl,3,5-pyridinedicarboxylate (DDC) increased the mass of ALA synthase in intestine and kidney of chick embryos. Furthermore, the molecular mass of the subunit of the enzyme in those tissues appeared identical with that of liver ALA synthase. Using a synthetic oligonucleotide complementary to ALA synthase mRNA, we determined by solution hybridization and Northern-blot analyses that AIA and DDC also increased the concentrations of ALA synthase mRNA in intestine and kidney and that testosterone elevated the concentration of the RNA in kidney. In analyses of RNA obtained from chick-embryo liver, intestine, kidney, heart, brain and lung, the probe bound primarily in each case to a single 2.3 kb RNA. Finally, the haem precursors ALA and FeCl3, when injected together into the fluid surrounding embryos, inhibited both the elevations in ALA synthase mass and RNA concentration brought about by porphyrogenic agents in liver, kidney and intestine. Thus the results indicated that: (1) certain porphyrogenic agents increased ALA synthase mass and RNA in chick-embryo intestine and kidney, in addition to liver; (2) ALA and FeCl3 inhibited the elevations; and (3) the sizes of ALA synthase's subunit as well as the enzyme's mRNA appeared identical, in each case, in all tissues examined.

Regulation of the stability of chicken embryo liver delta-aminolevulinate synthase mRNA by hemin.

The effect of hemin on the stability of delta-aminolevulinate (ALA) synthase mRNA was investigated in primary cultures of chicken embryo hepatocytes. Hepatocytes were first incubated with allylisopropylacetamide (AIA) to increase the starting level of ALA synthase mRNA, and then the cells were incubated with alpha-amanitin (1 microgram/ml) to block further transcription of ALA synthase RNA. The rates of depletion of the enzyme's mRNA were then determined by liquid hybridization analyses in cells incubated with or without hemin (10 microM) in the culture medium. The analyses indicated that hemin increased the rate of ALA synthase mRNA degradation. The half-life of the messenger in hepatocytes incubated with hemin was 80 min compared with 220 min in cells incubated without additional hemin in the culture medium. The results thus indicated a novel mechanism by which hemin modulated the biogenesis of ALA synthase in liver.

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme.

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of biogenesis of liver delta-aminolevulinate synthase: effects of structural modifications of heme on the enzyme's RNA.

1. The aim of this study was to determine the effects of several metallo-porphyrins, derived by modifications of heme, on the concentration delta-aminolevulinate (ALA) synthase RNA in hepatocytes. 2. Primary cultures of chick embryo hepatocytes were incubated with allylisopropylacetamide (AIA) for 5 hr in the presence and absence of each metallo-porphyrin (10 microM). At the end of each incubation, total RNA was isolated from the cells and analyzed for ALA synthase-specific RNA by solution hybridization. 3. The concentration of ALA synthase RNA increased 7.3 fold in hepatocytes incubated with AIA alone. The AIA-induced elevations in the enzyme's RNA were blocked partially and equally in cells. incubated with zinc- or with iron-protoporphyrin IX. The block was greater in cells incubated with cobalt-protoporphyrin IX. 4. Modifications of the side chains of the porphyrin ring at positions 2 and 4, giving mesoporphyrin IX and deuteroporphyrin IX, changed the effectiveness of the iron- and the cobalt-porphyrins to limit the AIA-induced increase in ALA synthase RNA. The modifications did not affect the capacities of the zinc-porphyrins to inhibit the rise in RNA. 5. In conclusion, the effect of a given metallo-porphyrin on liver ALA synthase RNA following side chain modification depended on the coordinated metal.

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase.

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.

Properties of chicken erythrocyte delta-aminolevulinate synthase.

A procedure is described for preparing a fraction highly enriched for chicken blood delta-aminolevulinate synthase (ALA-S) using animals recovering from acetylphenylhydrazine-induced anemia. 1. Blood cells collected from chickens recovering from anemia were disrupted by nitrogen cavitation, and the mitochondrial fraction was prepared from the cell homogenates. ALA-S was released then from mitochondria by sonication and isolated by a procedure involving gel filtration chromatography on Sephadex G-150, fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephacel, and preparative isoelectric focusing. 2. Electrophoretic analyses under denaturing conditions indicated that the final ALA-S preparation was particularly enriched from a 62,200 Da polypeptide. The enzyme eluted from Sephadex G-200 with an equivalent molecular weight of 115,000; this suggested that active ALA-S was a dimer. 3. ALA-S was most active in the pH range of 7.0-8.0, with an apparent KM of 13 microM for succinyl-CoA and of 4.0 mM for glycine. The activity was inhibited 50% by 30 microM hemin.

Maturation of embryonic chick liver delta-aminolevulinate synthase: precursor pools and regulation by intra-cellularly produced heme.

1. Immunoblot analyses were carried out to determine the relative distributions of delta-aminolevulinate synthase (ALA synthase) in mitochondrial and cytosol fractions prepared from embryos at different times after injections with allylisopropylacetamide (AIA). 2. The results indicated that the molecular mass of mature ALA synthase (Mr 65,000) increased with time in mitochondria. 3. At no time was the precursor form (Mr 75,000) of the enzyme detected either in mitochondria or in the cytosol. 4. In primary cultures of hepatocytes, where the increased production of ALA synthase had been induced with AIA, addition of delta-aminolevulinic acid (ALA) and Fe2(SO4)3 into the culture medium completely blocked the processing of the precursor form of the enzyme. 5. On the other hand, the addition of ALA together with deferoxamine mesylate into the medium had no detectable effect on the maturation of ALA synthase in the hepatocytes. 6. The results indicated: first, that upon induction of porphyria the pools of pre-ALA synthase in liver are relatively low in chick embryos when compared with those in other organisms; and second, that increased heme production by the hepatocytes caused the inhibition of processing of the precursor form of ALA synthase.

Biogenesis of embryonic chick liver delta-aminolevulinate synthase: regulation of the level of mRNA by hemin.

The effects of hemin on the concentration of the mRNA for delta-aminolevulinate synthase (ALA synthase) and on the association of the messenger with polysomes were investigated in primary cultures of embryonic chick hepatocytes incubated with allylisopropylacetamide (AIA). A synthetic 24-mer DNA complementary to ALA synthase mRNA was used to determine by solution hybridization the effects of AIA and of AIA plus hemin on the ALA synthase-specific RNA sequences in the cells. The results indicated that ALA synthase mRNA concentrations increased significantly in hepatocytes incubated for 5 h with AIA (0.075 mg/ml), and that hemin in the medium (2 or 10 microM) blocked the increase in the messenger. When delta-aminolevulinic acid (ALA) and FeCl3 were added into the culture medium (1 mM and 5 microM, respectively), the increase in ALA synthase mRNA brought on by AIA was also inhibited. Neither ALA nor FeCl3, when individually added to the cultures, was as effective as the combination of the two. The results with ALA + FeCl3 suggested that stimulation of intracellular production of heme was also effective in blocking the increase in ALA synthase mRNA caused by AIA. Finally, the distributions of ALA synthase mRNA were compared in polysomes isolated from hepatocytes which had been incubated with AIA for 5 h in the presence and absence of 10 microM hemin in the medium. Although a drop was detected in the concentration of ALA synthase mRNA in polysomes from hepatocytes incubated with hemin for 30 min, the decrease was explained by the effect of hemin on the mRNA concentration in the cells.

Regulation of production of embryonic chick liver delta-aminolevulinate synthase: effects of testosterone and of hemin on the mRNA of the enzyme.

The effects of testosterone and of hemin on the concentration of the mRNA of embryonic chick liver ALA synthase were investigated. Using cDNA-RNA liquid hybridization analyses, we determined that testosterone, when injected into the fluid surrounding chick embryos, caused a dose-dependent increase in the concentration of ALA synthase mRNA in liver. Similarly, addition of testosterone (5 micrograms/ml) or of 75 micrograms/ml of allylisopropylacetamide (AIA) into the medium of chick embryo hepatocytes maintained in culture caused an increase in the concentration of ALA synthase mRNA. Hemin (2 or 5 microM), when added to the culture medium, inhibited the elevations of ALA synthase mRNA concentration brought on by testosterone and by AIA.

Biogenesis of liver delta-aminolevulinate synthase. The role of cAMP in the induction.

In primary cultures of chick embryo hepatocytes pulse labeled with [35S]methionine, immunochemical analyses indicated that adenosine 3':5'-cyclic monophosphate (cAMP) did not affect either the rate of production or the maturation of delta-aminolevulinate synthase (ALA synthase). In addition, allylisopropylacetamide caused a slight drop in intracellular cAMP while testosterone caused the levels of cAMP to rise to 260% of the basal levels measured in hepatocytes in culture. Thus the results of this study did not indicate a direct short-term role for cAMP in the regulation of production of ALA synthase.

Biogenesis of mitochondrial proteins. Regulation of production of delta-aminolaevulinate synthase by haemin in embryonic-chick liver.

The effect of haemin on the biogenesis of delta-aminolaevulinate synthase (ALA synthase) was investigated in primary cultures of embryonic-chick liver. The activity of the enzyme and the amount of the enzyme detected by 'immune-blotting' were determined in hepatocytes incubated with the porphyrogenic agent allylisopropylacetamide. The results of these studies indicated that the loss in ALA synthase activity in cells incubated in the presence of haemin (10 microM) was roughly proportional to a loss in the immune-reactive mass of the enzyme. Haemin was as effective as cycloheximide in causing depletion of ALA synthase in hepatocytes. We had previously established that haemin blocked the maturation of the precursor of ALA synthase [Ades (1983) Biochem. Biophys. Res. Commun. 110, 42-47]. From results reported in the present paper on analyses of immune-precipitated ALA synthase after pulse-labelling with [35S]methionine in the presence and in the absence of haemin, we determined that the inhibition of processing of pre-ALA synthase in cells by haemin was concentration-dependent. A concentration of 2 microM in the culture medium blocked the processing of pre-ALA synthase by 50% in hepatocytes. We also determined that, after inhibition of its maturation by haemin, pre-ALA synthase turned-over with a half-time of 30 min; on the other hand, mature ALA synthase turned-over with a half-time of 120 min.

Biogenesis of mitochondrial proteins regulation of maturation of delta-aminolevulinate synthase by hemin.

The effect of hemin on the biogenesis of delta-aminolevulinate (ALA) synthase was examined in primary cultures of chick embryo hepatocytes. Hemin (0.010 mM) in the culture medium significantly inhibited the induction of ALA synthase activity in hepatocytes exposed to the porphyrogenic agent allylisopropylacetamide. In hepatocytes pulse-labeled with [35S]methionine for 45 min in the presence and absence of hemin, it was determined by immunechemical analyses that hemin blocked the processing of the precursor of ALA synthase.

Isolation of the mature subunit of delta-aminolaevulinate synthase from embryonic chick liver.

We presented evidence indicating that the established procedure for purifying delta-aminolaevulinate (ALA) synthase from embryonic-chick liver yielded an enzyme with a partially degraded subunit of molecular weight 51000 [Ades & Harpe (1981) J. Biol. Chem. 256, 9329-9333]. We now report the purification from livers of porphyric embryos of a preparation of ALA synthase which consisted primarily of a 63000-Da polypeptide and a component migrating as a smear of polypeptides with a minimum molecular weight of 52 000. Neither component could be recovered from liver mitochondria of normal embryos, where the amounts of ALA synthase were relatively low. The 52 000-Da component had been established to be the partially degraded subunit of the enzyme. Peptide-mapping analyses indicated that the 63 000- and the 52 000-Da components possessed significant structural homologies, and it was concluded that the 63 000-Da polypeptide represented the mature subunit of ALA synthase.