Purification and characterization of palmitoyl-CoA ligase from rat brain microsomes

We have previously shown the existence of two separate enzymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA in rat brain microsomal membranes (1). Palmitoyl-CoA ligase activity was solubilized from brain microsomal membranes with 0.3% Triton X-100 and purified 93-fold by a combination of protein purification techniques. The Km values for the substrates palmitic acid, CoASH and ATP were 11.7 microM, 5.88 microM and 3.77 mM respectively. During activation of palmitic acid ATP is hydrolyzed to AMP and pyrophosphate, as evidenced by the inhibition of this activation by 5 mM concentrations of AMP, pyrophosphate or AMP and pyrophosphate to 70%, 60% and 85% respectively. The divalent metal ion Mg2+ was required for activity; its replacement with Mn2+ resulted in a 35% decrease in activity. Palmitoyl-CoA ligase activity was inhibited by the addition of oleic or stearic acids whereas addition of lignoceric acid or behenic acid had no effect. This supports our previous observation that palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes in rat brain microsomal membranes.     

Isoprenoid enzyme systems of silkworm. II. Formation of the juvenile hormone skeletons by farnesyl pyrophosphate synthetase II

Comparative substrate specificities of farnesyl pyrophosphate synthetases I and II purified from larvae of silkworm, Bombyx mori, were studied by use of the possible biosynthetic intermediates of juvenile hormones in the insect. In the presence of Mn2+ ions farnesyl pyrophosphate synthetase II showed higher activity than synthetase I and the corresponding enzyme from pig liver with the following substrate homologues: (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, (2E,6Z)-3,7-dimethyl-2,6-nonadienyl-, and (2E,6Z)-3-ethyl-7-methyl-2,6-nonadienyl pyrophosphate. When (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, and isopentenyl pyrophosphate were mixed and incubated with farnesyl pyrophosphate synthetase II, (2E,6E,10Z)-3,11-dimethyl-7-ethyl-2,6,10-tridecatrienyl-, (2E,6E,10Z)-3,7,11-trimethyl-2,6,10-tridecatrienyl, and a trace amount of (2E,6E,10Z)-3,7-diethyl-11-methyl-2,6,10-tridecatrienyl pyrophosphate, whose carbon skeletons were the same as those of juvenile hormone I, II, and O, respectively, were formed. (Z)-3-Methyl-2-pentenyl pyrophosphate was produced from 3-ethyl-3-butenyl pyrophosphate as a single product by the action of silkworm isopentenyl pyrophosphate isomerase, though the enzyme activity was much lower with this substrate than with the usual substrate, isopentenyl pyrophosphate.     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Formation of dolichyl pyrophosphate N-acetylglucosaminyl intermediates

Liver microsomes from pig embryos synthesized dolichyl pyrophosphate N-acetylglucosamine and converted it to dolichyl pyrophosphate N,N'-diacetylchitobiose. N-acetylglucosaminyl transferase activity towards dolichol was about 2-fold greater in microsomes from embryonic liver than in microsomes from adult liver. A maximum level of conversion of dolichyl pyrophosphate N-acetylglucosamine to dolichyl pyrophosphate N,N'-diacetylchitobiose was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM). The level of dolichyl phosphate, assessed by the level of dolichyl pyrophosphate N-acetylglucosamine synthesis was 2-fold higher in microsomes from embryonic liver than that in microsomes from adult liver. Tunicamycin (1 microgram/ml) inhibited completely the formation of dolichyl pyrophosphate N-acetyl-glucosamine in embryonic liver microsomes, while the inhibitory effect of UMP (1 mM) was about 70%.     

Substrate specificity of cis-prenyltransferase in rat liver microsomes.

Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with Km values of 24 and 5 microM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent Km of 36 microM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyl 12-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.     

Characterization and distribution of cis-prenyl transferase participating in liver microsomal polyisoprenoid biosynthesis.

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.     

Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction

Isoprene formation in a rat liver cytosolic fraction is shown to be increased 146-fold by acid treatment. This acid catalysis is dependent upon prior incubation of the cytosolic fraction with DL-mevalonate and is stimulated when the incubation also contains ATP. Formation of isoprene proceeds linearly through 5 h of acid treatment and is nearly complete at 10 h. These results suggest that the acid-catalyzed isoprene formation arises from the decomposition of dimethylallyl pyrophosphate via a carbonium ion mechanism. Chemical model studies using 3-methyl-2-buten-1-ol and 3-methyl-3-buten-1-ol (the alcohols corresponding to dimethylallyl pyrophosphate and isopentenyl pyrophosphate, respectively) confirm this hypothesis. At a pH less than or equal to 1, an 85% decomposition of 3-methyl-2-buten-1-ol to isoprene occurred after 24 h, while 3% of 3-methyl-3-buten-1-ol was converted to isoprene under identical conditions and time. It is concluded that the predominant immediate precursor of isoprene is dimethylallyl pyrophosphate and at low pH the ultimate fate of dimethylallyl pyrophosphate is complete conversion to isoprene. These conclusions have important biochemical and methodological implications.     

The partial purification and properties of a phytoene synthesizing enzyme system.

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent K_m for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn²⁺, but not for NADP⁺. At a concentration of 2 mm, NADP⁺ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at ?20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.     

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.     

Hepatic accumulation of pyrophosphate during acetate metabolism

Accumulation of pyrophosphate induced by acetate administration was investigated in rat liver in situ and in perfused rat liver. Intraperitoneal injection of acetate into rats increased the pyrophosphate concentration in the liver to about 2 mumol/g liver, which was 200 times that in control liver. Perfusion of liver with acetate alone did not result in accumulation of pyrophosphate. However, the further addition of a Ca2+-mobilizing hormone, such as noradrenaline or angiotensin II, together with glucagon to the perfusion medium containing 1 mM acetate caused accumulation of pyrophosphate to a similar level to that observed in vivo. Acetate, glucagon and a Ca2+-mobilizing hormone were all required for accumulation of pyrophosphate in perfused liver. Omission of Ca2+ from the perfusion medium or addition of a Ca2+-antagonist reduced the accumulation significantly. The two kinds of hormones, glucagon and an alpha-agonist, either singly or in combination, did not affect the rate of acetate utilization. These results show that liver cells accumulate a large amount of pyrophosphate during acetate metabolism at high intracellular levels of Ca2+ that can be realized by the synergistic actions of the two kinds of hormones.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Substrate selectivity of squalene synthetase.

Six 1-3H-labeled analogues of farnesyl pyrophosphate have been studied as potential substrates for yeast and rat liver squalene synthetases: 2-methylfarnesyl pyrophosphate (4), 3-demethylfarnesyl pyrophosphate (5), 7,11-dimethyl-3-ethyl-2,6,10-dodecatrienyl pyrophosphate (6), 6,7,10,11-tetrahydrofarnesyl pyrophosphate (7), 4-methylthiofarnesyl pyrophosphate (8), and 4-fluorofarnesyl pyrophosphate (9). Analogues 4 and 5 are enzymatically incorporated into 11-methylsqualene (10) and 10-demethylsqualene (11), respectively, even if no farnesyl pyrophosphate is added to the incubations. None of the other analogues gives nonpolar products with either the yeast or liver enzymes. No tritium is enzymatically released to the medium from any of the analogues, indicating that they are not accepted at the first (proton exchanging) site. The data rule out formation of dead-end presqualene pyrophosphate products with analogues as first, but not as second, substrates. Implications of these results for the enzyme active-site topology and mechanism are discussed.     

Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.     

Quinolinic Acid Phosphoribosyltransferase in Rat Brain

Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 +/- 0.30 microM for quinolinic acid and Km = 65.13 +/- 13.74 microM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 +/- 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 +/- 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a greater than 20-fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippo-campus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve-ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at -80 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis

2-Oxoglutarate decarboxylase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. It had a molecular weight of 250,000 and consisted of four identical subunits of molecular weight 62,000. The enzyme was specific for 2-oxoglutarate, but not for other 2-oxo acids such as pyruvate and oxalacetate. Thiamin pyrophosphate and MgCl2 were required for maximum activity. The Km values of the enzyme for 2-oxoglutarate, thiamin pyrophosphate, and MgCl2 were 330, 56, and 93 microM, respectively. 2-Mercaptoethanol and NADP+ augmented significantly the enzyme activity. The amino acid composition and amino acid sequence of the amino-terminal region of 2-oxoglutarate decarboxylase were determined. On ouchterlony double-immunodiffusion gels, the anti-2-oxoglutarate decarboxylase antibody gave sharp precipitin lines against the mitochondrial fraction of E. gracilis and the purified 2-oxoglutarate decarboxylase, but not against pyruvate decarboxylase from Saccharomyces cerevisiae. On Immunoblots of the crude extract of Euglena, the antibody recognized two polypeptides whose molecular weights were 62,000 and 65,000, respectively. The polypeptide with the molecular weight of 62,000 was found only in mitochondrial fractions. In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in Euglena cells.     

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta.

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6P and beta-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate: glucose phosphotransferase, and undetectable [U-14C]G6P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate/pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Purine metabolism in high- and low-uric acid lines of chickens: hypoxanthine/guanine phosphoribosyltransferase activities

The activity of hypoxanthine/guanine phosphoribosyltransferase (HGPRT) was examined in the livers and kidneys of two genetic lines of chickens selected for different plasma uric acid levels. Previous work demonstrated that the high-uric acid line (HUA) had significantly greater de novo uric acid synthesis rates in kidney tissue compared to the low-uric acid line (LUA). In addition, phosphoribosylpyrophosphate (PRPP) synthetase and xanthine dehydrogenase activities in livers and kidneys were significantly higher in the HUA compared to the LUA line. PRPP pool sizes were also significantly higher in both livers and kidneys of HUA birds. HGPRT activities in livers of HUA birds were significantly (P less than 0.05) greater than in LUA birds. The mean value of liver HGPRT was 7.36 +/- 0.25 pmole inosine-5'-monophosphate (IMP) and 6.05 +/- 0.27 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA lines. There were no significant differences (P greater than 0.05) in kidney HGPRT activities between the two groups. The mean value of kidney HGPRT was 52.87 +/- 1.62 pmole IMP and 50.72 +/- 1.62 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA line. Elevated liver HGPRT may serve to enhance the regeneration of PRPP in the HUA liver. Elevated liver PRPP synthetase and PRPP pool size suggest an increased flux through the de novo purine biosynthetic pathway in HUA birds. The resulting additional pyrophosphate from the glutamine PRPP amidotransferase reaction would stimulate recovery of PRPP and spare the system from a substantial loss of energy.     

Biosynthesis of monoterpenes: partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis.

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage (Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl₂ (1 mm). ZnCl₂ was nearly as effective as MnCl₂, and MgCl₂ could substitute for MnCl₂ only at tenfold higher concentrations. The apparent K_m and V of the enzyme were 10^?5m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10^?4m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10^?5m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.     

The purification of 3,3-dimethylallyl- and geranyl-transferase and of isopentenyl pyrophosphate isomerase from pig liver

The enzyme catalysing the synthesis of farnesyl pyrophosphate from dimethylallyl pyrophosphate and isopentenyl pyrophosphate, or from geranyl pyrophosphate and isopentenyl pyrophosphate, has been purified 100-fold from homogenates of pig liver. The enzyme has optimum pH 7.9 and requires Mg(2+) as activator in preference to Mn(2+); it is inhibited by iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate and phosphate ions in addition to the products of the reaction, inorganic pyrophosphate and farnesyl pyrophosphate. From product-inhibition studies of the geranyltransferase reaction, the order of addition of substrates to and release of products from the enzyme has been deduced: geranyl pyrophosphate combines with the enzyme first, followed by isopentenyl pyrophosphate. Farnesyl pyrophosphate dissociates from the enzyme before inorganic pyrophosphate. The existence of isopentenyl pyrophosphate isomerase in liver is confirmed. Methods for the preparation of the pyrophosphate esters of isopentenol, 3,3-dimethylallyl alcohol, geraniol and farnesol are also described.