The effect of hypolipidemic drugs WY14643 and DH990, and lysophospholipids on the metabolism of oleate in plants

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [¹⁴C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [¹⁴C]oleate or [¹⁴C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [¹⁴C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

Acylation of lysophosphatidylcholine in bovine heart muscle microsomes: purification and kinetic properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase

Bovine heart muscle microsomes rapidly convert lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in the presence of oleoyl-CoA. Both substrates are incorporated into the product, although the rate of incorporation of radiolabel into PC from 1-[14C]palmitoyl-LPC was approximately threefold higher than the rate of incorporation from [14C]oleoyl-CoA. Furthermore, the rate of incorporation of radiolabel from [14C]LPC was stimulated fivefold by the presence of oleoyl-CoA. These results demonstrate the presence of both acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) and an LPC:LPC transacylase (EC 3.1.1.5) in microsomes. Separation of the two enzymatic activities and purification of the acyltransferase was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The isolated acyltransferase was a single species of 64,000 Da as judged by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The substrate specificity of the enzyme was studied by using a series of lysophospholipids as acyl acceptors and acyl-CoA derivatives as acyl donors. The enzyme was catalytically active with LPC as acyl acceptor but displayed little or no activity with lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine. Of the LPC derivatives tested, the highest activity was obtained with 1-palmitoyl-LPC. Wider specificity was exhibited for the nature of the acyl donor, for which arachidonoyl-CoA, linoleoyl-CoA, and oleoyl-CoA were highly active substrates. These properties of the acyltransferase are in accord with a role of the enzyme in determining the composition of PC in myocardium.     

Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed

The acyl coenzyme A (CoA) preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm (Butia capitata Becc.), maize (Zea mays L.), and rapeseed (Brassica napus L.) was tested. Each microsomal preparation was incubated with [¹⁴C-U]-glycerol-3-phosphate and either lauroyl CoA, oleoyl CoA, or erucoyl CoA, and the ¹⁴C-lipid products were separated and quantitated. In the presence of oleoyl CoA, the microsomes from each of the three species produced lysophosphatidic acid, phosphatidic acid, diacylglycerol, and triacylglycerol with kinetics consistent with the operation of the glycerol phosphate pathway. In the presence of erucoyl CoA, the microsomes from all the three species did not produce di- or tri-acyl lipids. In the presence of lauroyl CoA, only the microsomes from palm, but not those from maize or rapeseed, synthesized di- and tri-acyl lipids. This lack of reactivity of lauroyl CoA was also observed in the microsomes from maturing castor bean, peanut, and soybean. In maize seed and rapeseed, but not palm seed, the kinetics of labeling suggest that lauroyl and erucoyl moieties of the acyl CoAs were incorporated into lysophosphatidic acid but failed to enter into phosphatidic acid and thus the subsequent lipid products. We propose that the high degree of acyl specificity of lysophosphatidyl acyltransferase is the blocking step in the synthesis of triacylglycerols using lauroyl CoA or erucoyl CoA. The significance of the findings in seed oil biotechnology is discussed.     

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

Phospholipid acylation by mouse sciatic nerve microsomes.

The partition of 0.3 nmol of [1-14C]oleoyl-CoA in the microsomes (10 micrograms proteins) from mouse sciatic nerves is unaffected by the presence of lysophospholipids and is about 45% of the total oleoyl-CoA (77% of the acylglycerophosphocholine partition in the membrane). The concentration of both oleoyl-CoA and acylglycerophosphocholine is over 1 mM in the membrane. There is a selective acyl transfer from acyl-CoA to lysolipid acceptors (oleoyl greater than myristoyl, palmitoyl, stearoyl much greater than eicosanoyl greater than docosanoyl, tetracosanoyl). The exogenous acyl acceptors are acylglycerophosphocholine and acylglycerophosphoinositol and to a lesser extent acylglycerophosphoethanolamine, but not acylglycerophosphoserine. A PC formation from acylGPC in the absence of exogenous acyl donors or from oleoyl-CoA in the absence of exogenous acyl acceptor was also observed.     

Remodelling of triacylglycerols in microsomal preparations from developing castor bean (Ricinus communis L.) endosperm

Microsomal preparations from developing castor bean (Ricinus communis L.) endosperm catalyzed remodelling of in-situ-formed triacylglycerol (TAG) species. Castor bean microsomal membranes synthesized [¹⁴C]TAGs from either glycerol 3-phosphate and [¹⁴C]ricinoleoyl-CoA or [¹⁴C]glycerol 3-phosphate and ricinoleoyl-CoA. Upon repelleting and subsequent incubation of the microsomes a redistribution occurred of both the [¹⁴C]glycerol and [¹⁴C]ricinoleoyl moieties of the in-situ-synthesized [¹⁴C]TAGs. Radioactivity was transferred from TAG species with three (3HO-TAG) or two (2HO-TAG)ricinoleoyl groups into species with two or one (HO-TAG) ricinoleoyl groups. Mass analysis of the lipid and fatty acid movements in the membranes showed that a net synthesis of TAGs with no, one and two ricinoleoyl groups occurred at the expense of 3HO-TAG and polar lipids. Thus, the non-hydroxylated acyl groups from polar lipids were used in the remodelling of TAGs. In-vivo feeding of [¹⁴C]ricinoleic acid to slices of castor bean endosperm demonstrated the presence of two radioactive pools of TAGs one in the oil bodies, which was rich in [¹⁴C]3HO-TAG, and one associated with the microsomal membranes, which was dominated by radioactive 1HO-TAG and 2HO-TAG. The microsomal TAG pool was remodelled in vivo in a similar way as in the in-vitro experiments with microsomal membranes. Received: 8 November 1996 / Accepted: 5 February 1997     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Growth,carbohydrates and associated invertase and amylase activities in castor bean and maize as affected by metribuzin and NaCl

Growth parameters (leaf area, length of shoot and root, water content and dry matter accumulation), contents of reducing sugars and saccharose as well as activities of α- and β-amylases of castor bean and maize seedlings and adult plants supplemented with 0.5 μg g^?1 and 2.5 μg g^?1 of metribuzin either alone or in combination with 50 μg g^?1 NaCl, were increased significantly whereas at high concentrations (5 and 10 μg g^?1) of herbicide, an opposite response was apparent. On the other hand, polysaccharide content and invertase activity of castor bean and maize seedlings and adult plants were significantly decreased in response to low concentrations (0.5 and 2.5 μg g^?1) of metribuzin and increased significantly at high concentrations (5 and 10 μg g^?1) of the herbicide either alone or in combination with 50 μg g^?1 NaCl. Total carbohydrate contents of castor bean and maize seedlings and whole plants treated with herbicide either alone or in combination with NaCl did not change significantly. Growth parameters, carbohydrate fractions contents and activities of enzymes in both castor bean and maize seedlings and whole plants treated with herbicide alone were consistently higher than those values detected in plants treated with herbicide in combination with NaCl.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.     

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl₂. The K_m for UDP-glucose was determined as 27 micromolar, and the K_m for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [³²P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Transfer of phospholipids from microsomes to mitochondria in germinating castor bean endosperm

¹⁴C-labeled microsomes were prepared by feeding [1-¹⁴ C]acetateto endosperm tissues from 4-day-old seedlings of castor beanseeds and incubated with unlabeled mitochondria from the sametissues. The loss of ¹⁴C-lipids from the microsomes was accompaniedby an increase of ¹⁴C-lipids in the mitochondria. The additionof 105,000?g supernatant and also pH 5.1-treated supernatant,both of which had been prepared from castor bean endospermsat the same stage, markedly enhanced the lipid transfer frommicrosomes to mitochondria. The activity in this fraction wasprecipitated by ammonium sulfate and lost with trypsin or heattreatment. The transfer of lipids was limited to phospholipids.Thus, it is concluded that in castor bean endosperms, phospholipidsare transferred from the endoplasmic reticulum to the mitochondriaby a phospholipid-exchange protein contained in the cytosol. (Received August 8, 1977; )     

Cytosol-stimulated remodeling of phosphatidylcholine in rat lung microsomes

When 600 × g supernatants of 10% (w/v) rat lung homogenates were incubated with CDP[Me-¹⁴C]choline, both saturated and unsaturated species of phosphatidylcholine were formed from endogenous diacylglycerols. The percentage radioactivity in the disaturated species of total phosphatidylcholine increased with time from 12% after 5 min to 30% after 60 min incubation. In similar experiments with 20000 × g supernatants, the increase in the disaturated species of microsomal phosphatidylcholine was from 25 to 37% over the same time period. In incubations of isolated microsomes in buffer, the percent of ¹⁴C label in disaturated phosphatidylcholine remained constant at a level of 25%. To investigate a possible role of cytosolic factor(s) in the increase in the percentage of disaturated phosphatidylcholine with time, microsomes were prelabeled by incubation in buffer with CDP[Me-¹⁴C]choline to give a fixed ratio of radioactive saturated and unsaturated phosphatidylcholine species. When the reisolated microsomes were incubated in buffer, the distribution of radioactivity over saturated and unsaturated species remained constant. In contrast, incubation of prelabeled microsomes in the presence of cytosol caused an increase in the percent radioactivity in saturated phosphatidylcholines from a starting value of 18 to 30% after 60 min incubation, while leaving total phosphatidylcholine radioactivity unaffected. These results indicate a remodeling of phosphatidylcholine under the influence of a cytosolic factor(s). Evidence is presented that suggests that Ca²⁺-independent cytosolic phospholipase A₂ activity as well as a microsomal ATP-independent CoA-mediated acyltransferase activity might contribute to this remodeling. The cytosol donates the necessary CoA for this acyl transfer as well as saturated acyl-CoA for the reacylation of lysophosphatidylcholine.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: evidence for the presence of cytochrome P-450.

The aryl hydrocarbon hydroxylase (AHH) enzyme from the fungus Cunninghamella bainieri has been characterized. It is NADPH dependent and exhibits a pH optimum near 7.8. It is inhibited by CO, SKF 525-A, and metyrapone, but cyanide shows no inhibitory effect. These data, together with the pattern of inhibition and stimulation shown by metal ions, suggest that the fungal AHH activity is due to a cytochrome P-450. About 25% of the hydroxylase activity remains in the supernatant while the remainder precipitates after centrifugation at 100,00g for 2.5 h. The 100,000g supernatant was further fractionated by (NH₄)₂SO₄ precipitation. A NADPH-dependent cytochrome c reductase is concentrated mainly in the 100,000g supernatant, and a cytochrome c oxidase is present mainly in the 100,000g pellet. The cytochrome c reductase is essential for AHH activity as shown by the inhibition of AHH activity with cytochrome c and dichloroindophenol. Solubilization of a portion of the 100,000g pellet in aqueous digitonin followed by dithionite reduction and addition of CO resulted in the observation of a maximum absorbance at 450 nm characteristic of cytochrome P-450.     

Characterization of an enzyme from Phaseolus vulgaris seeds which hydroxylates GAi to GA8

Hydroxylation of gibberellin-[³H] A₁ (GA₁-[³H]) to GA₈-[³H] by the 95000 g supernatant fluid from imbibed bean seeds required Fe²⁺ or Fe³⁺ and O₂ but was insensitive to CO. The hydroxylating enzyme has a sedimentation coefficient of 4·5 S, and was precipitated by (NH₄)₂SO₄ at 35–60% saturation. This hydroxylase was specific for GA₁ and did not hydroxylate either pseudo-GA₁-[³H] or 16-ketoGA₁-[³H]. Virtually all hydroxylase activity was localized in the cotyledons.