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At least 91 records · Page 5Linked to original sources
PubMed1977

Effects of detergents on the properties of 4-hydroxybenzoate. Polyprenyl transferase and the specificity of the polyprenyl pyrophosphate synthetic system in mitochondria.

The properties of 4-hydroxybenzoate:polyprenyl transferase and the system synthesizing polyprenyl pyrophosphate have been studied in mitochondria from rat and guinea pig livers. With solanesyl pyrophosphate and 4-hydroxybenzoate as substrates the formation of 3-nonaprenyl-4-hydroxybenzoate was linear with time, concentration of protein, and concentration of solanesyl pyrophosphate. Solanesyl monophosphate is inactive as a substrate and is noninhibitory. Conversion of solanesyl monophosphate to the pyrophosphate could not be detected. Detergents such as Triton X-100, Tween-80, and sodium deoxycholate activated the enzyme in mitochondria which were aged by freezing at -20 degrees C for periods ranging from 1 h to several days. Maximum activation also required Mg2+. In agreement with previous observation the effect of Mg2+ and Triton X-100 on fresh mitochondria was quite variable; however, activation with aged preparations was very consistent. Treatment with TritonX-100 causes al alteration in the biosynthetic pattern of rat liver mitochondria so that rather than nonaprenyl, decaprenyl, pyrophosphate is preferentially made in the presence of solanesyl pyrophosphate and isopentenyl pyrophosphate. In the presence of Triton X-100 and added pool of solanesyl pyrophosphate appears to exert a feedback inhibition on the incorporation of isopentenyl pyrophosphate into solanesyl pyrophosphate. In the case of guinea pig liver mitochondria a different pattern is observed with Triton X-100 in contrast to the rat. The de novo formation of decaprenyl pyrophosphate from isopentenyl pyrophosphate appears to be inhibited by Triton X-100, but the synthesis of decaprenyl pyrophosphate from isopentenyl pyrophosphate and nonaprenyl pyrophosphate is not inhibited. The data also indicate that in guinea pig liver in a system synthesizing decaprenyl pyrophosphate from isopentenyl pyrophosphate, there does not appear to be a detectable pool of nonaprenyl pyrophosphate. These results show that detergents can affect the specificity of the mitochondrial system synthesizing polyprenyl pyrophosphates.

Animals↗
PubMed1981

Geranylgeranyl pyrophosphate synthetase lacking geranyl-transferring activity from Micrococcus luteus.

Geranyl pyrophosphate synthetase, which catalyzes the condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate to give geranyl pyrophosphate, was purified 490-fold from Micrococcus luteus extracts by DEAE-Sephadex, hydroxylapatite, and Sephadex G-100 column chromatography. The enzyme has a pH optimum at 7.7 and the molecular weight was estimated to be 70,000 by Sephadex gel filtration. The Km values for isopentenyl pyrophosphate and dimethylallyl pyrophosphate were 8 microM and 62 microM, respectively. The enzyme required Mg2+ for maximum activity. Tween 80 showed a stimulative effect whereas Triton X-100 was rather inhibitory on the enzyme activity. Inorganic pyrophosphate and iodoacetamide were both potent inhibitors of the enzyme. The purified enzyme fraction was also capable of catalyzing the synthesis of geranylgeranyl pyrophosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate, but lacked geranyl-transferring activity. The catalytic activities of geranylgeranyl pyrophosphate synthesis and geranyl pyrophosphate synthesis were affected differently by iodoacetamide and Triton X-100. This enzyme fraction may be a mixture of two enzymes, geranyl pyrophosphate synthetase and geranylgeranyl pyrophosphate synthetase catalyzing the reactions of C5 greater than C10 and C15 greater than C20, respectively, or a single enzyme with two independent catalytic sites responsible for the C5 greater than C10 and C15 greater than C20 reactions. In any case, the existence of a new geranylgeranyl pyrophosphate synthetase different from the known geranylgeranyl pyrophosphate synthetase catalyzing the continuous condensation reaction of C5 greater than C10 greater than C15 greater than C20 was demonstrated.

Alkyl and Aryl Transferases↗
PubMed1989

Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis).

Cell-free homogenates from sage (Salvia officinalis) leaves convert dimethylallyl pyrophosphate and isopentenyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [1-14C]dimethylallyl pyrophosphate and [1-3H]isopentenyl pyrophosphate was verified as [14C,1-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradative studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the C10 precursor for the production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases.

Cell-Free System↗
PubMed1996

Calcium pyrophosphate dihydrate crystal deposition disease as a cause of lumbar canal stenosis.

STUDY DESIGN: This study measured the incidence of calcium pyrophosphate dihydrate crystal deposition in specimens of ligamenta flava in consecutive patients undergoing decompressive laminectomy between 1984 and 1991. The results were compared to determine the difference between calcium pyrophosphate dihydrate-negative patients with lumbar canal spinal stenosis. OBJECTIVES: The results were compared with cadaver specimens and literature values to determine if calcium pyrophosphate dihydrate crystal deposition disease contributes to the thickening of the ligamentum flavum and thereby contributes to spinal stenosis. SUMMARY OF BACKGROUND DATA: Calcium pyrophosphate dihydrate crystal deposition disease has been described in the axial skeleton. Hypertrophy of the ligamentum flavum has been suggested to contribute to stenosis. The association of calcium pyrophosphate dihydrate disease and hypertrophied ligamenta flava has not been fully defined nor linked to neurologic symptoms and signs. METHODS: The incidence of calcium pyrophosphate dihydrate crystal deposition in specimens of ligamenta flava obtained from four groups was measured: specimens obtained during surgery from 102 consecutive patients undergoing decompression laminectomy between 1984 and 1991, 47 additional pathologic specimens of ligamentum flavum tested between 1984 and 1991, 222 calcium pyrophosphate dihydrate-positive Pathology Department specimens collected between 1980 and 1991, and, as control specimens from 20 cadavers. The associated patient histories were reviewed for the first two groups; no histories were available for the cadaver group. RESULTS: The incidence of calcium pyrophosphate dihydrate crystal deposition was 24.5% in the ligamentum flavum among the surgical patients, 31% among the Pathology Department specimens, 33.8% among the calcium pyrophosphate dihydrate-positive Pathology Department specimens, and 5% among the cadavers. No associated medical conditions with calcium pyrophosphate dihydrate crystal deposition were found among the medical histories. Patients with the symptoms of spinal stenosis who were also calcium pyrophosphate dihydrate-negative patients with symptoms of less than 6 months' and less than 24 months' duration (P < 0.001). Except for time to presentation, calcium pyrophosphate dihydrate-negative patients had similar signs and symptoms of lumbar canal spinal stenosis. Having previous spine surgery did not produce a statistically significant risk of having calcium pyrophosphate dihydrate crystal deposition. No specific laboratory tests were found to be of predictive value. CONCLUSIONS: These findings suggest that calcium pyrophosphate dihydrate crystal deposition may indeed be associated with the thickening of the ligamentum flavum, if so, patients may benefit from medical treatment before undergoing surgical treatment of lumbar canal spinal stenosis.

Aged↗
PubMed1976

Transition state analogs for thiamin pyrophosphate-dependent enzymes.

Many of the transition states that are formed from thiamin pyrophosphate in enzymic reactions are expected to have structures in which the thiazolium ring of thiamin pyrophosphate has lost most of its positive charge. We have synthesized thiamin thiazolone pyrophosphate from the unphosphorylated compound. The sulphur-containing ring of thiamin thiazolone pyrophosphate is uncharged, and thus the compound resembles these transition states. In agreement with the prediction from the transition state theory of reaction rates, thiamin thiazolone pyrophosphate binds to Escherichia coli pyruvate dehydrogenase complex (EC 1.2.7.1) much more strongly than thiamin pyrophosphate itself. An upper limit for the value of the dissociation constant, calculated from the extent of inactivation of the enzyme by a low concentration of thiamin thiazolone pyrophosphate, is 5 X 10(-10) M at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. The dissociation constant for thiamin pyrophosphate under similar conditions is about 10(-5) M. The kinetics of inactivation of pyruvate dehydrogenase complex by thiamin thiazolone pyrophosphate are first order with respect to both enzyme and thiamin thiazolone pyrophosphate; the value of the second order rate constant is 5.7 X 10(5) M-1 min-1 at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. An analysis of the decrease in the rates of inactivation caused by thiamin pyrophosphate indicates that thiamin thiazolone pyrophosphate binds at the coenzyme sites. We have also synthesized thiamin thiothiazolone pyrophosphate and obtained very similar results with this compound.

Alkaline Phosphatase↗
PubMed1990

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes.

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.

Acyclic Monoterpenes↗