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B V Milborrow

Publications and source records attributed to B V Milborrow.

At least 19 recordsLinked to original sources
PubMed2001

The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis.

The pathway of biosynthesis of abscisic acid (ABA) can be considered to comprise three stages: (i) early reactions in which small phosphorylated intermediates are assembled as precursors of (ii) intermediate reactions which begin with the formation of the uncyclized C40 carotenoid phytoene and end with the cleavage of 9'-cis-neoxanthin (iii) to form xanthoxal, the C15 skeleton of ABA. The final phase comprising C15 intermediates is not yet completely defined, but the evidence suggests that xanthoxal is first oxidized to xanthoxic acid by a molybdenum-containing aldehyde oxidase and this is defective in the aba3 mutant of Arabidopsis and present in a 1-fold acetone precipitate of bean leaf proteins. This oxidation precludes the involvement of AB-aldehyde as an intermediate. The oxidation of the 4'-hydroxyl group to the ketone and the isomerization of the 1',2'-epoxy group to the 1'-hydroxy-2'-ene may be brought about by one enzyme which is defective in the aba2 mutant and is present in the 3-fold acetone fraction of bean leaves. Isopentenyl diphosphate (IPP) is now known to be derived by the pyruvate-triose (Methyl Erythritol Phosphate, MEP) pathway in chloroplasts. (14C)IPP is incorporated into ABA by washed, intact chloroplasts of spinach leaves, but (14C)mevalonate is not, consequently, all three phases of biosynthesis of ABA occur within chloroplasts. The incorporation of labelled mevalonate into ABA by avocado fruit and orange peel is interpreted as uptake of IPP made in the cytoplasm, where it is the normal precursor of sterols, and incorporated into carotenoids after uptake by a carrier in the chloroplast envelope. An alternative bypass pathway becomes more important in aldehyde oxidase mutants, which may explain why so many wilty mutants have been found with this defect. The C-1 alcohol group is oxidized, possibly by a mono-oxygenase, to give the C-1 carboxyl of ABA. The 2-cis double bond of ABA is essential for its biological activity but it is not known how the relevant trans bond in neoxanthin is isomerized.

Abscisic Acid↗
PubMed1994

beta-Carotene-15,15'-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure.

beta-Carotene-15,15'-dioxygenase (EC 1.13.11.21; beta-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15'-14C2]- or [15,15'-3H2] beta-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled beta-carotene and beta-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining beta-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled apo-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol beta-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More beta-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The beta-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formed/mg protein per h. The formation of a lipid-protein aggregate containing the beta-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal scrapings were homogenized in buffer at a proportion of 1:4 (w/v).

Animals↗
PubMed1992

The Uptake of (+)-S- and (-)-R-Abscisic Acid by Suspension Culture Cells of Hopbush (Dodonaea viscosa).

The uptake of (+)-S- and (-)-R-abscisic acid (ABA) by suspension culture cells of hopbush (Dodonaea viscosa L. Jacqu.) was followed over a range of temperatures, pH values, and time intervals. The natural (+)-S-ABA was taken up about five times faster than the unnatural (-)-R-ABA. Each 10 degrees C rise in temperature from 1 to 31 degrees C increased the rate of uptake (Q(10)) of (+)-S-ABA about 2.2-fold, whereas that of the (-)-R increased with a Q(10) of 1.4. (+)-ABA was taken into the cells by a saturable carrier, but (-)-ABA and both enantiomers of 2-trans-ABA were not; they appeared to enter by passive diffusion. The uptake of (+)-ABA was linear over the first 8 hours but concentrations within the cells decreased after 2 hours to remain constant after 4 hours as rapid metabolism was induced. Electron microscopy of thin sections of the cells, combined with a stereological analysis of their shape, showed that the vacuoles comprised 80% of the cell volume and the cytoplasm plus nucleus comprised 20%. There were no photosynthetically active plastids in the cells. Concentrations of the endogenous ABA in the cytoplasm (pH 7.32) and vacuoles (pH 5.88) were calculated by applying the Henderson-Hasselbalch equation (ABA pK(a) 4.7) so that, provided no active metabolic redistribution occurred, the concentration in the cytoplasm was 7.9 micromolar and that in the vacuole was 0.3 micromolar. In vivo pH was measured by (31)P nuclear magnetic resonance spectroscopy.

Journal Article↗
PubMed1984

Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography.

Attempts to resolve the enantiomers of racemic abscisic acid (ABA) by high-performance liquid chromatography on a chiral stationary-phase column were unsuccessful. However, reduction of RS-methyl ABA (RS-Me-ABA) with sodium borohydride generates a new chiral centre and one of the two isomeric products, the RS-Me-1',4'-cis-diol of ABA, was separated into its enantiomers by high-performance liquid chromatography on an optically active Pirkle column. High-performance liquid chromatography on a mu Bondapak C18 column separated the metabolites and conjugates of [2-14C]ABA fed to tomato shoots. The resolution method was used to measure the relative proportions of R and S enantiomers in the free acid liberated from conjugates of ABA.

Abscisic Acid↗
PubMed1984

The conformation of abscisic acid by n.m.r. and a revision of the proposed mechanism for cyclization during its biosynthesis.

The n.m.r. spectrum of abscisic acid (ABA) formed from [1,2-13C2]acetate by the fungus Cercospora rosicola shows 13C-13C coupling between C-6' (41.7 p.p.m.; 36 Hz) and the downfield 6'-methyl group (6'-Me) (24.3 p.p.m, 36 Hz). This 6'-Me, therefore, is derived from C-3' of mevalonate [Bennett, Norman & Maier (1981) Phytochemistry 20, 2343-2344]. An i.n.e.p.t. (insensitive nuclei enhanced by polarization transfer) pulse sequence demonstrated that the downfield 13C signal is produced by the 6'-Me that gives rise to the upfield 1H 6'-Me signal (23.1 d). The absolute configuration of this, the equatorial 6'-Me group, was determined as 6'-pro-R by decoupling and n.O.e. (nuclear-Overhauser-enhancement) experiments at 300 MHz using ABA, ABA in which the axial 6'-pro-S 5'-hydrogen atom had been exchanged with 2H in NaO2H and the 1',4'-cis- and 1',4'-trans-diols formed from these samples. The configuration at C-1' and at C-6' are now compatible with a chair-folded intermediate during cyclization, as proposed for beta- and epsilon-rings of carotenoids. ABA in solution exists, as in the crystalline form, with the ring in a pseudo-chair conformation. The side chain is axial and the C-3 Me and the C-5 hydrogen atoms are predominantly cis(Z).

Abscisic Acid↗
PubMed1981

Stereochemistry of beta-, gamma-, and epsilon-ring formation in bacterial C50.

Cell-free systems from Corynebacterium poinsettiae and Micrococcus luteus incorporated labeled mevalonic acids into acyclic C40 and cyclic C50 carotenoids. When (3R,4R)-[2-14C,4-3H1]mevalonate was used as substrate, the 14C:3H ratios of C.p.450 and sarcinaxanthin showed that the hydrogen atoms at C-2 of both carotenoids, and that at C-6 of sarcinaxanthin, are derived from the 4-pro-R position of mevalonate. The 14C:3H ratios of C.p.450 and sarcinaxanthin synthesized from (2RS,3R)-[2-14C,2-3H2]mevalonate showed that both hydrogen atoms of C-4 are derived from those at C-2 of mevalonate. These results exclude epsilon- and beta-rings as precursors of the gamma-ring. They also exclude the interconversion of the epsilon- and beta-rings. Sarcinaxanthin samples synthesized from (3R,4R)-[2-14C,4-3H1]- and (2RS,3R)-[2-14C,2-3H2]mevalonate by a cell-free system from M. luteus were found to undergo isomerization in strong alkali. The major product of isomerization (85%) was decaprenoxanthin (epsilon-ring) with the beta-ring C.p.450 present in small amounts (3% yield). The 14C:3H ratios of these isomerization products were consistent with the loss of one C-4 hydrogen atom from each epsilon-ring of the former and one C-6 hydrogen atom from each beta-ring of the latter.

Carbon Radioisotopes↗
PubMed1981

Stereochemistry of allene biosynthesis and the formation of the acetylenic carotenoid diadinoxanthin and peridinin (C37) from neoxanthin.

Intact cells of the alga Amphidinium carterae (Dinophyceae), and a cell-free system prepared from it, incorporated 14C, 3H-labelled mevalonate into lycopene, beta, beta-carotene, zeaxanthin, neoxanthin, diadinoxanthin and peridinin. The 14C/3H ratios of zeaxanthin, neoxanthin and diadinoxanthin formed from (2RS,3R)-[2-14C,2-3H2]mevalonate show that a hydrogen atom from C-2 of mevalonate is retained in the allene at C-8, and also at C-12 of peridinin. (3R,4R + 3S,4S)-[2-14C,4-3H1]Mevalonate gave 14C/3H ratios in peridinin which show that C-14 is lost. The three carbon atoms excised during the formation of the C37 carotenoid peridinin are C-13, C-14 and C-20 of neoxanthin.

Acetylene↗
PubMed1980

Retention of the 4-pro-R hydrogen atom of mevalonate at C-2,2' of bacterioruberin in Halobacterium halobium.

Intact cells of Halobacterium halobium fed with (3R,4R)-[2-14C,4-3H1]mevalonic acid were found to incorporate label into acyclic C40 and C50 carotenoids, of which bacterioruberin was the most abundant. The 14C/3H ratios of the isolated carotenoids demonstrated that the 4-pro-R hydrogen atom of mevalonic acid was retained at the C-2 and C-2' positions of bacterioruberin. Diphenylamine was found to inhibit the production of bacterioruberin.

Carbon Radioisotopes↗
PubMed1972

Stereochemical aspects of the formation of double bonds in abscisic acid.

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.

Amides↗
PubMed1970

Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants.

(+/-)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-(14)C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-(3)H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (+/-)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-(14)C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by (18)O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.

Carbon Isotopes↗