首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
The stem content of diterpene resin acids (rosin) increases dramatically following wounding of grand fir (Abies grandis) saplings, but the level of monoterpene olefins (turpentine) in the stem decreases following injury, in spite of a significant increase in monoterpene cyclase (synthase) activity. However, this observation was explained when rapid evaporative losses of the volatile monoterpenes from the wound site was demonstrated by trapping experiments, a finding consistent with a role of turpentine as a solvent for the mobilization and deposition of rosin to seal the injury. Mature forest trees responded to stem wounding by the enhancement of monoterpene cyclization capacity in a manner similar to 2-year-old grand fir saplings raised in the greenhouse. Light and water stresses greatly reduced the constitutive level of monoterpene cyclase activity and abolished the wound-induced response. The diminution in monoterpene biosynthetic capacity was correlated with a dramatic decrease in cyclase protein as demonstrated by immunoblotting. Relief of stress conditions resulted in the restoration of cyclase activity (both constitutive and wound induced) to control levels. The results of these experiments indicate that grand fir saplings are a suitable model for studies of the regulation of defensive oleoresinosis in conifers.  相似文献   

2.
Monoterpene cyclase activity is greatly increased in grand fir (Abies grandis) sapling stems in response to wounding and the composition of the cyclic olefin mixture generated differs from that produced constitutively as determined by radio gas-liquid chromatography. Cell-free extracts from wounded stems and from non-wounded controls were systematically compared for monoterpene cyclase activities following partial purification and separation of these enzymes by anion-exchange chromatography (Mono Q FPLC) and native PAGE. The increase in monoterpene cyclase activity following wounding represents both the apparent enhancement of constitutive cyclase activities and the appearance of novel cyclization enzymes that are absent in nonwounded controls. A pinene cyclase was shown to be the major wound-inducible enzyme directly responsible for oleoresin monoterpene formation and was tentatively identified as a 62-kDa protein by SDS-PAGE.  相似文献   

3.
Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determined by monitoring the activity of monoterpene and diterpene cyclases, as well as two cytochrome P450-dependent diterpenoid hydroxylases involved in the formation of ([mdash])-abietic acid, the principal resin acid of this species. The activity of these enzymes reaches maximum levels that are 5- to 100-fold higher than those of nowwounded control stems 10 d after wounding and this is followed by a synchronous decline. The increase in biosynthetic activity is consequently followed by the accumulation of a viscous mass of resin acids, with the loss of the volatile monoterpenes, at the site of injury. The observed coordinate induction of monoterpene olefin and abietic acid bio-synthesis and the results of oleoresin analysis are consistent with the role of the volatile monoterpenes as a solvent for the mobilization and deposition of resin acids at the wound site to seal the injury with a rosin barrier after the evaporation of the turpentine. The last step of resin acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase that is not inducible by wounding but seemingly is expressed constitutively at a high level. In vivo [14C]acetate feeding and resin analysis indicate that this enzyme is not efficiently coupled to the earlier steps of the pathway.  相似文献   

4.
Studies were conducted to determine whether herbivore-induced synthesis of monoterpenes occurs in the needles of ponderosa pine (Pinus ponderosa Lawson), lodgepole pine (P. contorta Douglas var. latifolia Engelmann), white fir (Abies concolor Lindl. and Gordon) and Engelmann spruce [Picea engelmanii (Parry) Engelm.]. In the needles of all species except Engelmann spruce, simulated herbivory significantly induced the activity of monoterpene cyclases 4–8 days after wounding. In ponderosa pine, real herbivory by last-instar tiger moth larvae (Halisdota ingens Hy. Edwards: Lepidoptera) induced a significantly larger response (4.5-fold increase in monoterpene cyclase activity) than did simulated herbivory (2.5-fold increase). To our knowledge, this is the first report of herbivore-induced increases in monoterpene synthesis in needle tissue. Despite this increase in monoterpene synthesis, we observed no significant increase in total monoterpene pool size in wounded needles compared to controls. Large increases in the rate of monoterpene volatilization were observed in response to wounding. We conclude that the volatile losses caused by tissue damage compensate for herbivore-induced monoterpene synthesis, resulting in no change in pool size. Tiger moth larvae consume ponderosa pine needles in a pattern that begins at the tip and proceeds downward to midway along the needle, at which point they move to an undamaged needle. Constitutive monoterpene concentrations and monoterpene cyclase activities were highest in the lower half of ponderosa pine needles. The monoterpene profile also differed between the upper and lower needle halves, the lower half possessing an additional one to four monoterpene forms. We propose that the increasing gradient in monoterpene concentrations and number of monoterpenes along the needle from tip to base deters feeding beyond the midway point and provides time for the induction of increased cyclase activity and production of new monoterpenes. The induction of new monoterpene synthesis may have a role in replacing monoterpenes lost through damage-induced volatilization and preventing extreme compromise of the constitutive defense system. Received: 4 June 1997 / Accepted: 2 December 1997  相似文献   

5.
Levels of monoterpene cyclase activity were determined in extracts from wounded and unwounded saplings of 10 conifer species to assess whether oleoresin biosynthesis is induced by stem wounding. Species of Abies and Picea, with low to moderate levels of constitutive monoterpene cyclase activity, exhibited a five- to 15-fold increase in cyclase activity 7 days after wounding relative to unwounded controls. In contrast, species of genera such as Pinus, with high levels of constitutive cyclase activity, did not significantly respond to wounding by alteration in the level of cyclase activity. The highest fold increase in monoterpene cyclase activity was consistently observed in Abies grandis, and the time-course of induction of activity following stem wounding in this species demonstrated a threefold increase at 2 days relative to unwounded controls, rising to a maximum increase in the response at 9 days (greater than 10-fold) followed by an apparent decline. The wound response was localized, and both bark (phloem) and wood (xylem) tissues displayed increased cyclase activity at the wound site. The magnitude of the increase in cyclase activity was dependent on the severity of the wound.  相似文献   

6.
The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.  相似文献   

7.
To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10-2H3,1-3H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed.  相似文献   

8.
The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a pI value of about 5.0, as determined by isoelectric and chromatofocusing. SDS-PAGE showed the enzyme to be composed of two apparently identical subunits of Mr approximately 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg2+ (Km approximately 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The Km value for the substrate, farnesyl pyrophosphate, was 6.8 microM. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (alpha- and beta-patchoulene, alpha-bulnesene, and alpha-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.  相似文献   

9.
Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.  相似文献   

10.
The monoterpene cyclase, gamma-terpinene synthase, from Thymus vulgaris (thyme) leaves was purified to apparent homogeneity by isoelectric focusing and dye-ligand, anion-exchange, hydrophobic interaction, and gel permeation chromatography. The enzyme has a native molecular weight of 96,000 as determined by gel permeation chromatography, and exhibited a specific activity of 538 nmol/h.mg protein (turnover number of approximately 0.01/s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two apparently identical subunits of Mr approximately 55,000. The protein was very hydrophobic, and possessed a pI value of 4.85 as determined by isoelectric focusing. Maximum activity was observed at pH 6.8 in the presence of 20 mM Mg2+; 5 mM Mn2+ could support catalysis, albeit at a much lower rate. The Km value for the substrate, geranyl pyrophosphate, was 2.6 microM. Cyclase activity was inhibited by cysteine- and histidine-directed reagents. Purified gamma-terpinene synthase also possessed the ability to cyclize geranyl pyrophosphate to small amounts of alpha-thujene and to lesser quantities of myrcene, alpha-terpinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol, all of which appear to be coproducts of the reaction sequence leading to gamma-terpinene. In general properties, the gamma-terpinene synthase from thyme leaves resembles other monoterpene cyclases as well as sesquiterpene and diterpene cyclases.  相似文献   

11.
Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.  相似文献   

12.
Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.  相似文献   

13.
Humulene cyclase and caryophyllene cyclase, two enzymes which catalyze the cyclization of farnesyl pyrophosphate to the respective sesquiterpene olefins, have been partially purified from the supernatant fraction of a sage (Salvia officinalis) leaf epidermis extract and separated from each other by a combination of hydrophobic interaction, gel filtration, and ion-exchange chromatography. The molecular weight of both cyclases was estimated by gel filtration to be 57,000 and both cyclases exhibited a pH optimum of 6.5 and preferred Mg2+ (Km approximately 1.5 mM) as the required divalent metal cation. Both enzymes possessed a Km of about 1.7 microM for farnesyl pyrophosphate, were strongly inhibited by p-hydroxymercuribenzoate, and exhibited comparable sensitivities to a variety of other potential inhibitors. The properties of the two sesquiterpene olefin cyclases, which are the first from a higher plant source to be examined in detail, were very similar to each other and to other monoterpene, sesquiterpene, and diterpene cyclases previously described.  相似文献   

14.
Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool.  相似文献   

15.
(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.  相似文献   

16.
Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.  相似文献   

17.
Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the beta-NAD+ analogs 1,N6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.  相似文献   

18.
Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue.  相似文献   

19.
D Koesling  E B?hme  G Schultz 《FASEB journal》1991,5(13):2785-2791
Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.  相似文献   

20.
Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate:(+)-alpha-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage (Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号