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H Sagami

Publications and source records attributed to H Sagami.

At least 19 recordsLinked to original sources
PubMed1999

Human geranylgeranyl diphosphate synthase. cDNA cloning and expression.

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.

Alkyl and Aryl Transferases↗
PubMed1997

A partial deficiency of dehydrodolichol reduction is a cause of carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is a congenital disorder that involves the underglycosylation of N-glycosylated glycoproteins (Yamashita, K., Ideo, H., Ohkura, T., Fukushima, K., Yuasa, I., Ohno, K., and Takeshita, K. (1993) J. Biol. Chem. 268, 5783-5789). In an effort to further elucidate the biochemical basis of CDG syndrome type I in our patients, we investigated the defect in the multi-step pathway for biosynthesis of lipid-linked oligosaccharides (LLO) by the metabolic labeling method using [3H]glucosamine, [3H]mannose, and [3H]mevalonate. The LLO levels in synchronized cultures of fibroblasts from these patients were severalfold lower than those in control fibroblasts in the S phase, and the oligosaccharides released from LLO showed the same structural composition, Glc1 approximately 3.Man9.GlcNAc.GlcNAc, in the case of both the patients and controls. The amount of [3H]mannose incorporated into mannose 6-phosphate, mannose 1-phosphate, and GDP-mannose was greater in fibroblasts from these patients than in the control fibroblasts in the G1 period, although the ratios of these acidic mannose derivatives as indicated by the relative levels of radioactivity were the same for the two types of fibroblasts. Furthermore, upon metabolic labeling with [3H]mevalonate, the level of [3H]dehydrodolichol in fibroblasts from these patients increased in the S phase, and the levels of [3H]dolichol and [3H]dolichol-PP oligosaccharides concomitantly decreased, although the chain length distribution of the respective dolichols and dehydrodolichols was the same in the two types of fibroblasts. These results indicate that the conversion of dehydrodolichol to dolichol is partially defective in our patients and that the resulting loss of dolichol leads directly to underglycosylation.

Cells, Cultured↗
PubMed1997

Polyprenyl diphosphate synthases.

It is noteworthy that in spite of the similarity of the reactions catalyzed by these prenyltransferases, the modes of expression of catalytic function are surprisingly different, varying according to the chain length and stereochemistry of reaction products. These enzymes are summarized and classified into four groups, as shown in Figure 13. Short-chain prenyl diphosphates synthases such as FPP and GGPP synthases require no cofactor except divalent metal ions, Mg2+ or Mn2+, which are commonly required by all prenyl diphosphate synthases. Medium-chain prenyl diphosphate synthases, including the enzymes for the synthesis of all-E-HexPP and all-E-HepPP, are unusual because they each consist of two dissociable dissimilar protein components, neither of which has catalytic activity. The enzymes for the synthesis of long-chain all-E-prenyl diphosphates, including octaprenyl (C40), nonaprenyl-(C45), and decaprenyl (C50) diphosphates, require polyprenyl carrier proteins that remove polyprenyl products from the active sites of the enzymes to maintain efficient turnovers of catalysis. The enzymes responsible for Z-chain elongation include Z,E-nonaprenyl-(C45) and Z,E-undecaprenyl (C55) diphosphate synthases, which require a phospholipid. The classification of mammalian synthases seems to be fundamentally similar to that of bacterial synthases except that no medium-chain prenyl diphosphate synthases are included. The Z-prenyl diphosphate synthase in mammalian cells is dehydrodolichyl PP synthase, which catalyzes much longer chain elongations than do bacterial enzymes. Dehydrodolichyl PP synthase will be a major target of future studies in this field in view of its involvement in glycoprotein biosynthesis.

Alkyl and Aryl Transferases↗
PubMed1996

Enzymatic formation of dehydrodolichal and dolichal, new products related to yeast dolichol biosynthesis.

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C-isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.

Carbon Radioisotopes↗
PubMed1996

Proteolytic release of dehydrodolichyl diphosphate synthase from pig testis microsomes.

Pig testicular dehydrodolichyl diphosphate synthase was released in a soluble form out of microsomes by controlled proteolysis with trypsin or papain. Approximately 25% of the microsomal enzyme activity was recovered in the 115,000 x g supernatant fraction when the microsomes were treated with trypsin at 4 degrees C for 1 h. Similar proteolytic release of microsomal enzyme was also observed with the treatment with papain. The K(m), optimal pH, Mg2+ dependency, and ion strength dependency of the enzyme released by trypsin were similar to those of the microsomal enzyme. The microsomal enzyme was active even in the absence of detergents, while the released enzyme required detergents for activity. Gel filtration of the released enzyme gave a peak of dehydrodolichyl diphosphate synthase activity, which appeared between 150-kDa and 50-kDa molecular mass markers.

Alkyl and Aryl Transferases↗
PubMed1995

A novel type of protein modification by isoprenoid-derived materials. Diphytanylglycerylated proteins in Halobacteria.

Previous work from this laboratory has shown that a derivative of [3H]mevalonic acid is incorporated into a number of specific proteins in Halobacterium halobium and Halobacterium cutirubrum and that the major radioactive material released by treatment with methyl iodide was neither farnesyl nor geranylgeranyl compound, which have been generally accepted to be prenyl groups of a number of prenylated proteins found in eukaryotic cells, but an unknown compound (Sagami, H., Kikuchi, A., and Ogura, K. (1994) Biochem. Biophys. Res. Commun. 203, 972-978). In the current study, the unknown compound was prepared in a large amount from H. halobium cells and analyzed by reverse and normal phase high performance liquid chromatographies followed by mass spectrometry. The mass spectrum of this compound exhibited a parent ion peak (M+) at m/z 682, suggesting that it is a 1-methylthio-2,3-di-O-(3',7',11',15'-tetramethylhexadecyl)glycerol (diphytanylglyceryl methylthioether). Diphytanylglyceryl methyl thioether was chemically synthesized, and its mass fragmentation pattern was completely coincident with that of the mevalonic acid-derived material from H. halobium. These results indicate that Halobacteria contains specific proteins with a novel type of modification of a cysteine residue of the proteins with a diphytanylglyceryl group in thioether linkage.

Bacterial Proteins↗
PubMed1994

Novel isoprenoid modified proteins in Halobacteria.

Incorporation of [3H]mevalonic acid-derived materials into proteins was studied with extremely halophilic archaebacteria, Halobacterium halobium and Halobacterium cutirubrum. Several labeled proteins were detected on SDS-polyacrylamide gel electrophoresis followed by fluorography. The majority of the radioactive materials released from the labeled proteins by sulfonium salt cleavage moved with a mobility similar to that of a C85 polyprenol on reverse-phase thin-layer chromatography, and no radioactive farnesol was found on the chromatography. However, a weak but significant protein farnesyltransferase activity was detected in in vitro experiments with a combination of [3H]farnesyl diphosphate and Ras precursor protein.

Alkyl and Aryl Transferases↗
PubMed1994

Purification and properties of geranylgeranyl-diphosphate synthase from bovine brain.

Geranylgeranyl-diphosphate synthase was purified to homogeneity from bovine brain in a one-step procedure employing an affinity column. For the construction of the affinity column, a farnesyl diphosphate analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affi-Gel 10 via the amino group. The native enzyme appeared to be a homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were estimated to be 33, 0.80, and 0.74 microM, respectively. The Km value for isopentenyl diphosphate in the reaction with isopentenyl diphosphate and farnesyl diphosphate was 2 microM. The reaction velocities for the formation of geranylgeranyl diphosphate from dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate were in the ratio of 0.004:0.145:1. The intermediate farnesyl diphosphate was formed in the reaction with geranyl diphosphate as an allylic primer. Geranylgeranyl diphosphate acted as a competitive inhibitor against farnesyl diphosphate with an approximate Ki value of 1.2 microM in the condensation reaction of farnesyl diphosphate with isopentenyl diphosphate. Farnesyl-diphosphate synthase catalyzing the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate was also purified to homogeneity from the same organ by similar affinity chromatography using a geranyl diphosphate analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate, as a ligand. This enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that geranylgeranyl diphosphate, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from dimethylallyl diphosphate and isopentenyl diphosphate by the action of farnesyl-diphosphate synthase catalyzing the reaction of C5-->C15, followed by the action of geranylgeranyl-diphosphate synthase catalyzing a single reaction of C15-->C20, and that geranylgeranyl diphosphate can down-regulate its own synthesis through the inhibition of the geranylgeranyldiphosphate synthase action.

Alkyl and Aryl Transferases↗
PubMed1994

Phosphorylation of farnesol by a cell-free system from Botryococcus braunii.

Farnesol was incorporated into squalene as well as botryococcenes when the alcohol was fed to the culture of Botryococcus braunii B race strain. In in vitro experiments with a 10,000 x g supernatant of cell homogenate, squalene was synthesized from farnesyl diphosphate in the presence of NADPH or NADH, but botryococcenes were not synthesized under the same conditions. A 100,000 x g pelet fraction was able to phosphorylate farnesol to give its mono- and diphosphate esters in a CTP dependent manner.

Cytidine Triphosphate↗
PubMed1994

Purification of geranylgeranyl diphosphate synthase from bovine brain.

Geranylgeranyl diphosphate (GGPP) synthase was purified to homogeneity from bovine brain in a one-step affinity column procedure. For the construction of the affinity column, a farnesyl diphosphate (FPP) analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affigel 10 via the amino group. The native enzyme appeared to be homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and FPP were estimated to be 33 microM, 0.80 microM and 0.74 microM, respectively. The Km value for isopentenyl diphosphate (IPP) in the presence of both IPP and FPP mixture was 2 microM. The ratio of the reaction velocity for formation of GGPP from DMAPP, GPP or FPP was 0.004:0.145:1. The intermediate FPP was formed in the reaction with GPP as an allylic primer. FPP synthase catalyzing the formation of FPP from DMAPP and IPP was also purified to homogeneity from the same organ by a similar affinity chromatography procedure using a GPP analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate as a ligand. The enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that GGPP, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from DMAPP and IPP by the action of FPP synthase catalyzing the reactions C5-->C15 followed by the action of GGPP synthase catalyzing the reaction C15-->C20.

Alkyl and Aryl Transferases↗
PubMed1993

Formation of farnesal and 3-hydroxy-2,3-dihydrofarnesal from farnesol by protoplasts of Botryococcus braunii.

Farnesal and 3-hydroxy-2,3-dihydrofarnesal (3-hydroxy-3,7,11-trimethyl-6,10-dodecadiene-1-al) were formed from farnesol when the alcohol was incubated with the protoplast of Botryococcus braunii B race strain. This fact suggests the existence of farnesal hydratase in the alga. Feeding experiments showed that both farnesal and 3-hydroxy-2,3-dihydrofarnesal were efficiently incorporated into botryococcenes, triterpenoid hydrocarbons of the alga.

Carbon Radioisotopes↗
PubMed1993

Formation of dolichol from dehydrodolichol is catalyzed by NADPH-dependent reductase localized in microsomes of rat liver.

The alpha-saturation reaction involved in the biosynthesis of dolichol has been investigated with rat liver preparations. Under improved in vitro conditions with 10,000 x g supernatant of rat liver homogenates in the presence of NADPH at pH 8.0, dolichol was synthesized from isopentenyl diphosphate and Z,E,E-geranylgeranyl diphosphate. Neither dolichyl diphosphate nor dolichyl phosphate was detected. The chain length distribution of the dolicohol was the same as that of dehydrodolichyl products. In an assay system containing dehydrodolichol, dehydrodolichyl phosphate, or dehydrodolichyl diphosphate as a substrate, dehydrodolichol was predominantly converted into dolichol. The enzyme that catalyzes the conversion of dehydrodolichol to dolichol was localized in microsomes. The reductase activity was stimulated 9-fold by the addition of a 100,000 x g soluble fraction. The reductase had an opimal pH at 8.0. These results indicate that dolichol is formed from dehydrodolichol in rat liver microsomes. The formation of dolichol from dehydrodolichol was also catalyzed by 10,000 x g supernatant of rat or pig testis homogenates.

Animals↗
PubMed1993

Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.

When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.

Animals↗
PubMed1993

Geranylgeranyl diphosphate synthase catalyzing the single condensation between isopentenyl diphosphate and farnesyl diphosphate.

Geranylgeranyl diphosphate synthase was purified 191-fold from bovine brain by Mono Q column chromatography followed by preparative isoelectric focusing electrophoresis and Superose 12 gel filtration. The synthase had a pI value at 6.0, and it was made free of farnesyl diphosphate synthase, the pI of which was 5.1. The partially purified enzyme catalyzed the formation of geranylgeranyl diphosphate from isopentenyl diphosphate and farnesyl diphosphate with the Km values for isopentenyl diphosphate and farnesyl diphosphate being 14 and 0.8 microM, respectively. Dimethylallyl diphosphate and geranyl diphosphate were poor substrates with velocities of only 0.003 and 0.03, respectively, relative to that of farnesyl diphosphate. These results indicate that geranylgeranyl diphosphate synthase catalyzes a single condensation between isopentenyl diphosphate and farnesyl diphosphate and that farnesyl diphosphate is the common intermediate at the branch point for the synthesis of geranylgeranylated proteins as well as cholesterol, ubiquinone, dolichol, and farnesylated proteins. The enzyme required Mg2+ or Mn2+ for maximum activity. Octylglucoside showed a stimulatory effect on the enzyme activity.

Alkyl and Aryl Transferases↗
PubMed1992

Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate.

Geranylgeranyl diphosphate synthase from rat liver was separated from farnesyl diphosphate synthase, the most abundant and widely occurring prenyltransferase, by DEAE-Toyopearl column chromatography. The enzyme catalyzed the formation of E,E,E-geranylgeranyl diphosphate (V) from isopentenyl diphosphate (II) and dimethylallyl diphosphate (I), geranyl diphosphate (III), or farnesyl diphosphate (IV) with relative velocities of 0.09:0.15:1. 3-Azageranylgeranyl diphosphate (VII), designed as a transition-state analog for the geranylgeranyl diphosphate synthase reaction, was synthesized and found to act as a specific inhibitor for this synthase, but not for farnesyl diphosphate synthase. Diphosphate V and its Z,E,E-isomer (VI) also inhibited geranylgeranyl diphosphate synthase, but the effect was not as striking as that of the aza analog VII. Specific inhibition of geranylgeranyl diphosphate synthase by VII was also observed in experiments with 100,000g supernatants of rat brain and liver homogenates which contained isopentenyl diphosphate isomerase and prenyltransferases including farnesyl diphosphate synthase as well as geranylgeranyl diphosphate synthase. For farnesyl:protein transferase from rat brain, however, the aza compound did not show a stronger inhibitory effect than E,E,E-geranylgeranyl diphosphate.

Animals↗
PubMed1991

Formation of Z,E,E-geranylgeranyl diphosphate by rat liver microsomes.

Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.

Alkyl and Aryl Transferases↗