Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes

3-Hydroxy-3-methylglutaryl(HMG)-coenzyme A reductase purified from rat liver in the absence of protease inhibitors is composed of two distinct polypeptides of Mr = 51,000 and 52,500. Antibody raised to enzyme purified from rats fed a diet supplemented with cholestyramine and mevinolin inactivated HMG-CoA reductase. The antibody specifically precipitated a polypeptide of Mr = 94,000 from rat liver cells that had been previously incubated with [35S]methionine. The immunoprecipitation of the 35S-labeled polypeptide of Mr = 94,000 was prevented by addition of unlabeled pure HMG-CoA reductase (Mr = 51,000 and 52,500). Incubation of rat liver cells with mevalonolactone resulted in a decreased activity of HMG-CoA reductase and in a 40% decrease in the rate of incorporation of [35S]methionine into the immunoprecipitable reductase polypeptide of Mr = 94,000. In pulse-chase experiments, mevalonolactone enhanced the rate of degradation of the Mr = 94,000 polypeptide 3-fold. We propose that endogenous microsomal HMG-CoA reductase has a subunit of Mr = 94,000 and that the synthesis and degradation of this polypeptide are regulated by either mevalonolactone or, more likely, a product of mevalonolactone metabolism.     

Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes.

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.     

A new negatively regulated acute-phase phosphoprotein synthesized by rat hepatocytes.

The effect of acute inflammation on the production of the major phosphorylated protein (PP63) excreted by rat hepatocytes was investigated. Both intra- and extracellular forms of the protein labelled with [32P]Pi, [3H]fucose and [35S]methionine were immunoprecipitated with monospecific polyclonal antibodies, and relative rates of PP63 synthesis were measured. The hepatocytes of acutely inflamed rats produced and excreted 85% less 32P- and 3H-labelled PP63 than did control cells. This decreased amount of PP63 did not result from an impairment in the phosphorylation or glycosylation processes or from a blockade in excretion, but rather was found to be due to extensive shut-off in biosynthesis of the protein as measured by [35S]methionine incorporation. Thus PP63 would appear to represent a new negatively regulated acute-phase protein.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin.

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.     

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.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum.

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.     

Heat shock of Drosophila melanogaster induces the synthesis of new messenger RNAs and proteins.

The heat shock proteins, labelled in vivo with [35S]methionine, were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis and fingerprinted after tryptic digestion. Eight distinct heat shock polypeptides are characterized in this way. Heat shock messenger RNAs were isolated and partially purified. Assayed in vitro for protein synthesis, they were found to code for heat shock polypeptides. Some parameters of the kinetics of in vivo synthesis of the heat shock proteins are presented.     

Polypeptide synthesis by Rhizobium bacteroids and bacteria.

When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated [35S]-methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue. The labelled polypeptides were stable for several hours in pulse-chase experiments. Changes in the concentration of H+, K+ and Mg2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides. A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed [35S]methionine. Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared. Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase. Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns. The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.     

Comparison of ureaplasmas from sheep and goats with Ureaplasma diversum and U. urealyticum

Ureaplasmas isolated from sheep and goats were compared by immunofluorescence with antisera prepared in calves and by PAGE of polypeptides labelled by growth in the presence of [35S]methionine. The ovine and caprine strains constituted two groups defined by serology and polypeptide composition that were not related to the animal species from which they originated. Strains representing these two groups were compared with Ureaplasma urealyticum (human isolates) and U. diversum (bovine isolates). They could not be classified with either but were more similar to the U. diversum strains.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

A survey of differences between membrane polypeptides of transformed and nontransformed chick embryo fibroblasts

Plasma membrane-associated polypeptides of chick embryo fibroblasts and cells transformed by the Schmidt-Ruppin wild-type strain of Rous sarcoma virus and its temperature-sensitive tsNY68 mutant were compared by two-dimensional gel electrophoresis. Polypeptide and glycoprotein alterations were identified after incubation of cells with [35S]methionine and [3H]mannose and by staining of the gels with 125I-labeled concanavalin A and Coomassie brilliant blue. Polypeptides found to be consistently transformation-sensitive included a group of five polypeptides that were detected only by short-term labeling with methionine, fibronectin, a 180 kDa polypeptide with a pI of 5.6, a mannose-containing glycoprotein of 48 kDA and an unusually high pI of 8.4, and a 19 kDa polypeptide with a pI of approx. 4.5. Several of these polypeptides appear to be particularly interesting for further characterization.     

Mechanism(s) of heat killing: accumulation of nascent polypeptides in the nucleus?

To investigate the possibility that nascent polypeptides released from polysomes by heat shock accumulate in the nucleus, cells were pulse labeled with [35S]methionine for two minutes and heated immediately thereafter at 45.5 degrees C for 10 minutes. When isolated nuclei were subjected to gel electrophoresis and subsequently autoradiographed, heated nuclei exhibited an approximately 10-fold increase in radioactive polypeptides in comparison to nonheated controls. These nascent polypeptides were nonspecific molecules covering a wide range of molecular weights. It is plausible that the accumulation of polypeptides in the nucleus results in hyperthermic cytotoxicity. Therefore, we propose that a potential target for heat killing is within the nucleus, at sites where nascent polypeptides accumulate after heat shock.     

Protein synthesis in salivary glands of Drosophila hydei after experimental gene induction.

Several treatments, namely incubation at 37 degrees C, in the presence of arsenite, 2,4-dinitrophenol or vitamin B-6, or release from anaerobiosis induce the same set of puffs in the polythene chromosomes of salivary glands of Drosophila hydei. Analysis of changes in protein-synthetic patterns (as determined by radioautography of sodium dodecyl sulphate-gel electrophoretograms of extracts from [35S]methionine-labelled salivary glands) showed that concomitant with puff induction by these various treatments the same six strongly labelled polypeptide bands appeared. The amount of radioactive label in these peptides accounted for 25% of the total incorporation of [35S]methionine, except during incubation at 37 degrees C when it accounted for about 50%. The rate of synthesis of these peptides was maximal 1 h after the start of the puff-inducing treatment. The rate of decay of the rate of synthesis showed first-order kinetics both after removal of the puff-inducing stimulus or in the presence of actinomycin, with a half-life of approx. 4h.     

D-[35S(U)]inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes.

D-[35S(U)]myo-inositol 1,4,5-trisphosphorothioate [( 35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5-trisphosphate (InsP3), binds with high affinity (Kd 58.6 +/- 9.1 nM) to rat cerebellar membranes revealing a high density of specific binding sites (Bmax 21.5 +/- 2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]InsPS3 by InsP3. The results suggest that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.     

Disturbed assemble of the respiratory chain NADH: ubiquinone reductase (complex I) in citric-acid-accumulating Aspergillus niger strain B 60

Summary Previously we reported the selective loss of respiratory chain NADH:ubiquinone reductase (complex I) in the Aspergillus niger mutant B 60 during fermentation under citric-acid-accumulating conditions. The molecular basis underlying this complex I loss has been investigated. Complex I was isolated from A. niger wild-type and mutant B 60. No obvious differences in polypeptide composition and kinetic parameters were found between the two preparations. The mutant complex I, however, was very fragile and readily dissociated into subcomplexes when treated with salt. For studying the assembly of complex I, cells were labelled with [³⁵S]methionine and the flux of radioactive was followed through immunoprecipitable intermediates into the holo-complex. In the mutant, but not in the wild-type, large quantities of labelled complex I polypeptides were observed to accumulate transiently in an intermediate subcomplex. The possibility is discussed that in mutant B 60 the assembly of complex I is disturbed, leading to an unstable complex I.     

Biosynthesis of nonspecific lipid transfer protein (sterol carrier protein 2) on free polyribosomes as a larger precursor in rat liver

The biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S]methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S]polypeptides. The molecular mass of the former polypeptide was approximately 1.5 kDa larger than that of the purified mature nsLTP (13 kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5 kDa and converted to a mature form of 13 kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al. (1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5 kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13 kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils

Pre-treatment of human neutrophils with rGM-CSF resulted in a 3-fold increase in the rate of fMet-Leu-Phe stimulated reactive oxidant generation, as assessed by luminol- and lucigenin-chemiluminescence and O2- secretion. When blood-stream neutrophils were incubated in RPMI 1640 medium supplemented with [35S]methionine, both fMet-Leu-Phe (0.1 microM) and gamma-interferon (100 U/ml) stimulated a 3-4-fold increased incorporation of label into TCA-precipitable material. Similarly, rGM-CSF (50 U/ml) also stimulated protein biosynthesis in bloodstream neutrophils, and newly labelled polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Two classes of polypeptides were visualised on these gels: the relative rate of labelling of one class changed very little upon rGM-CSF treatment whereas the relative rate of labelling of a second group increased 3-12-fold.     

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[³⁵S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.