Role of cysteine in the dietary control of the expression of 3-phosphoglycerate dehydrogenase in rat liver.

Shifting rats to a protein-free, carbohydrate-rich diet, although not starvation, resulted in the appearance of mRNA for, and activity of, 3-phosphoglycerate dehydrogenase (3-PGDH) in liver as well as in a marked decrease in plasma cystine concentration. Refeeding with protein caused a 50% decrease in the mRNA in 8 h and its complete disappearance within 24 h, followed by a slower disappearance of the enzymic activity. Intraperitoneal administration of cysteine or methionine to protein-starved rats decreased the mRNA by 50-60% after 8 h. However, the repeated administration of cysteine failed to cause the complete disappearance of this mRNA in 24 h. In hepatocytes in primary culture, cysteine plus methionine and glucagon had, independently, an approx. 4-fold inhibitory effect on the abundance of the 3-PGDH mRNA and caused its almost complete disappearance when tested together. Insulin had an approx. 2-fold stimulatory effect, which was antagonized by cysteine plus methionine but was still apparent in the presence of glucagon. Nuclear run-on experiments and analysis of the stability of the mRNA with 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA polymerase II, suggested that the effect of cysteine plus methionine was due to destabilization of the mRNA, whereas the effect of glucagon was exerted on transcription. Cysteine, but not methionine, inhibited the accumulation of 3-PGDH mRNA in FTO2B hepatoma cells. In conclusion, the dietary control of the expression of the 3-PGDH gene in liver seems to involve the negative effects of cysteine and glucagon and the positive effect of insulin.

Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase.

Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human D- or L-hydroxyglutaric aciduria.

Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase).

A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses omicron-nitrophenyl acetate and alpha-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688-29696; Yan, Yang, and Parkinson (1995) Arch. Biochem. Biophys. 317, 222-234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.

Cloning and sequencing of rat liver carboxylesterase ES-3 (egasyn).

The cDNA of the rat carboxylesterase ES-3 encodes a polypeptide with 561 amino acid residues including a cleavable signal peptide at the N-terminus. The processed polypeptide shows over 90% sequence identity to mouse egasyn (ES-22); its calculated pI (5.5) matches the value determined for purified liver ES-3. The product expressed in COS cells migrates in native gels in the region of ES-3 and is similarly active on acetanilide. It is retained in the cells, as predicted from its C-terminus HTEL, and bears a single endo-H sensitive oligosaccharide chain. The nonglycosylated form expressed in the presence of tunicamycin is also intracellular, but substantially less active.

Topogenesis of carboxylesterases: a rat liver isoenzyme ending in -HTEHT-COOH is a secreted protein.

We have cloned a rat liver cDNA that encodes a carboxylesterase isoenzyme, as revealed by immunoprecipitation, cytochemical staining and inhibition by bis-p-nitrophenylphosphate of the product expressed in transfected COS cells. The predicted polypeptide ends in -HTEHT-COOH. The product is secreted by COS cells with a half-time of about 1 hour, after maturation of oligosaccharide chains in the Golgi complex. A variant ending in -HTEL-COOH is stable in the cells. This strengthens the existing evidence that the HXEL-COOH end signals proteins for retrieval from the secretory traffic in animal cells. The encoded enzyme still remains to be identified. It shows 98% homology to an esterase sequenced earlier (Takagi et al. 1988, J. Biochem. 104, 801-806; Long et al. 1988, Biochem. Biophys. Res. Commun. 156, 866-873); however it must be an enzyme from the serum, not from the microsomes.

The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum.

To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.

Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum.

A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5'-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5'-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5'-end non-coding region, 1695 bp of coding region and 212 bp of 3'-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts.

Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit. Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation. In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact. Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes. The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H. Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain. Metabolic labelling in hepatocytes cultured with [35S]methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium. This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell. Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not. The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum [Robbi & Beaufay (1986) Eur. J. Biochem. 158, 187-194; (1987) Biochem. J. 248, 545-550] indistinguishable from those occurring early in the synthesis of secretory proteins. Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

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.

Genetic identification of rat liver carboxylesterases isolated in different laboratories.

Six carboxylesterases previously isolated from rat liver microsomes, characterized in Brussels and in Kiel, were compared with genetically defined liver esterases of various reference strains using polyacrylamide gel electrophoresis and isoelectric focusing. The six liver carboxylesterases were identified as alloenzymic forms of ES-3, ES-4, ES-8/ES-10 and ES-15 according to the genetic nomenclature recommended by van Zutphen (Van Zutphen, L.F.M. (1983) Transplant. Proceed. 15, 1687-1688). The genetic and biochemical characteristics of the four isoenzymes are summarized, and their identity with several other drug-metabolizing esterases/amidases and lipases of rat liver endoplasmic reticulum is discussed.

Biosynthesis of rat-liver pI-5.0 esterases in cell-free systems and in cultured hepatocytes.

Rat liver esterases focusing at pH 5.0 (referred to below as pI-5.0 esterases) are structurally related glycoproteins which differ slightly in their mobility in sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). They reside in the lumen of the endoplasmic reticulum. We have studied their biosynthesis in cell-free systems programmed by total liver RNA, using sheep and rabbit antibodies to isolate the translation products related to these enzymes. Our results show that they are assembled as a precursor polypeptide chain (62 kDa) larger than the mature proteins. The pI-5.0 esterase mRNA could be extracted from bound but not free polysomes. Reticulocyte lysates supplemented with dog pancreas microsomes produced four esterase-related components in segregated form (61, 60, 58 and 56 kDa). The largest three correspond in electrophoretic mobility to the mature enzymes. They are glycoproteins that bind to concanavalin A, and can be reduced to the size of the shortest component by endo-beta-N-acetylglucosaminidase H (endo-H). Immunoprecipitation after biosynthetic labeling of the proteins in cultured hepatocytes also gave three glycosylated components that had the same mobility in SDS-PAGE as the mature enzymes. When tunicamycin was present in the culture medium, a single immunoprecipitable form was observed. Its apparent Mr was similar to that of the unglycosylated pI 5.0 esterase form synthesized in vitro in the presence of dog pancreas microsomes. Thus the biosynthesis of these esterases has characteristics in common with that of numerous secretory proteins, except for the rather large difference in size (approximately equal to 6 kDa) resulting from the proteolytic processing of their in-vitro-synthesized precursor.

Purification and characterization of various esterases from rat liver.

The major rat liver microsomal esterases acting on o-nitrophenylacetate with isoelectric points 5.0, 5.5, 6.1 and 6.4 were resolved by isoelectric focusing. Molecular weights were determined by sedimentation analysis in isokinetic gradients of sucrose and, after purification, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Their subunit molecular weights were between 57 000 and 60 000. They behaved as monomers except the pI-6.1 enzyme which behaved as a trimer. Esterases of pI 5.0, pI 6.1 and pI 6.4 behaved like glycoproteins of the polymannose type in the presence of 125I-labelled concanavalin A. Preparations of the pI-5.0 enzyme contained two esterases of highly homologous structure. Antibodies directed against this preparation did not inhibit but precipitated pI-5.0 esterase activity quantitatively. They did not react with the pI-6.1 and pI-6.4 esterases but precipitated several nonimmunologically related esterases. Two of these enzymes were inducible by phenobarbital. Total activity was very low in 3-day-old animals. Individual esterase activities rose at different rates during development; the enzyme focusing near pI 5.0 was about three times more active in adult females than in males. All microsomal esterases are located on the luminal side of the endoplasmic reticulum.

Peptide mapping of peroxisomal catalase and its precursor. Comparison to the primary wheat germ translation product.

To investigate possible structural modifications of catalase during its biogenesis and packaging into peroxisomes, we have labeled three species of catalase with [35S]methionine: the wheat germ cell-free translation product, the extraperoxisomal precursor made in vivo in rat liver, and mature peroxisomal catalase. These three species have identical mobilities in sodium dodecyl sulfate polyacrylamide gels, when analyzed separately or in mixtures. Tryptic digestion yields 10 [35S]Met-labeled peptides from each, which are indistinguishable when mapped in two dimensions by electrophoresis and chromatography. Partial proteolyses of the three species in sodium dodecyl sulfate gels yielded identical fragmentation patterns. The primary translation product of catalase was labeled with formyl[35S] methionine; its size was indistinguishable from the subunit of mature catalase. Its radioactivity appeared in dansyl methionine if and only if it was deformylated prior to dansylation. These results demonstrate that within the limits of the methods, catalase undergoes no covalent modification during its uptake into peroxisomes and its subsequent maturation to a tetrameric hemoprotein.

Synthesis of catalase in two cell-free protein-synthesizing systems and in rat liver.

Rat liver polysomal RNA was translated in the rabbit reticulocyte lysate and in the wheat germ cell-free protein-synthesizing systems, using [(35)S]methionine as label. The catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) that was synthesized was isolated by immunoprecipitation and characterized by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels followed by fluorography. The catalase made in both systems migrated more slowly during electrophoresis than did purified peroxisomal catalase. By comparison with standards of known molecular mass, the cell-free products were estimated to be about 4000 daltons larger than the purified enzyme. We also investigated the biosynthesis of catalase in vivo by injecting [(35)S]methionine into rats. The precursor of catalase known to be synthesized in liver and found in the high-speed supernatant 8 min later [Lazarow, P. B. & de Duve, C. (1973) J. Cell Biol. 59, 491-506] was isolated immunochemically. For comparison, 1-day-old completed catalase was immunoprecipitated from peroxisomes. The migrations in sodium dodecyl sulfate gels of the 8-min-old precursor and the subunit of the day-old enzyme were indistinguishable and approximately the same as the migration of the cell-free products. These results indicate that catalase's apparent size does not change when it enters peroxisomes but rather decreases during the chemical purification procedure.

Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods.

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b(5) and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-beta-glucosaminidase, beta-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.