The solubilization of carotenogenic enzymes of Phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-¹⁴C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(?) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(?) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at ?70°C.     

Carotenoids in fish IV. Salmonidae and Thymallidae from Polish waters

The author investigated the presence of various carotenoids in the Salmonidae and Thymallidae family by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

Abstract

- in the muscles of Salmo salar: astaxanthin (pure and ester), canthaxanthin, lutein and zeaxanthin.

- in the eggs of Salmo trutta m. trutta: β-carotene, iso- and zeaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Salmo trutta m. fario: β-carotene, canthaxanthin, 4-keto-4-hydroxy-β-carotene, astaxanthin (pure and ester), lutein, taraxanthin and astacene.

- in the eggs of Salmo gairdneri: β-carotene, γ-carotene (?), canthacanthin, isozeaxanthin, lutein and astaxanthin, and in the sperm Salmo gairdneri: β-carotene, γ-carotene (?), 4-keto-4-hydroxy-β-carotene, canthaxanthin, lutein and astaxanthin.

- in the eggs of Salvelinus fontinalis: ester astaxanthin, canthaxanthin, isozeaxanthin, lutein and astacene.

- in the eggs of Hucho hucho: β-carotene, tunaxanthin, lutein, taraxanthin and astaxanthin.

- in the eggs of Coregonus albula: β-carotene, 4-keto-4-hydroxy-β-carotene, ester astaxanthin, zeaxanthin, taraxanthin and astacene.

- in Coregonus lavaretus: a) in eggs: β-carotene, ester astaxanthin, canthaxanthin, iso- and zeaxanthin, lutein, taraxanthin and astacene b) in the sperm: canthaxanthin, 4-hydroxy-4-keto-β-carotene, isozeaxanthin and astaxanthin, and other organs: 4-hydroxy-α-carotene, canthaxanthin, tunaxanthin, monoepoxy lutein, lutein, iso- and zeaxanthin and astaxanthin.

- in the eggs of Coregonus peled: β-carotene, 4-keto-4-hydroxy-β-carotene, lutein, zeaxanthin, taraxanthin and astacene.

- in the eggs of Thymallus thymallus: β-carotene, tunaxanthin, lutein and astaxanthin.

     

In vitro carotenogenesis by wild type and mutants of Phycomyces blakesleeanus

Cell extracts from shake cultures of the wild type and six mutant strains of Phycomyces converted [2-¹⁴C] MVA into carotenes, squalene and prenyl phosphates. Oxygen was required for the desaturation of phytoene. When compared with the wild type, cells extracts of carB and carR mutants are much less effective in phytoene dehydrogenation and lycopene cyclization, respectively. This confirms previous conclusions about the biochemical functions of the carB and carR genes, which were based on genetic and in vivo studies. CarA strain mutants accumulate, in vivo, much less β-carotene than the wild type. This correlates with a 10-fold decrease in carotenogenesis in vitro. The addition of retinol to incubations of cell extracts of the wild type and C2 strains stimulated β-carotene formation. Both carB and carR mutants show enhanced total carotenogenic activities in vitro and the carS mutant shows a higher β-carotene-synthesizing activity than the wild type. It is suggested that the feed-back regulatory mechanism known to control this pathway operates at the level of enzyme synthesis.     

Investigations of carotenoids in some animals of the Adriatic Sea V. Echinodermata

The author investigated the carotenoids in the Echinodermata from Adriatic sea by means of columnar and thin-layer chromatography. The following carotenoids were identified:

- in Coscinasterias tenuispina: β-carotene, isocryptoxanthin lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin and asterinacid.

- in Marthasterias glacialis: β-carotene, echinenone, cryptoxanthin, lutein, lutein 5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin ester, astaxanthin and 3, 4-didehydro-α-carotene.

- in Paracentrotus lividus: β-carotene, echinenone, cryptothin, isocryptoxanthin, lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin, astaxanthin ester and asterinacid.

- in Sphaerechinus granularis: ,β-carotene, echinenone, cryptoxanthin, lutein, lutein-5, 6-epoxide, astaxanthin and guaraxanthin.

     

Genetic analysis of provitamin A carotenoid β-cryptoxanthin concentration and relationship with other carotenoids in maize grain (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Provitamin A (proVA) carotenoids are converted into retinol (vitamin A) in the human body, are the subject of human nutrition studies, and are targets for biofortification of staple crops. β-Carotene has been the principal target for enhancing levels of proVA. There is recent interest in enhancing the proVA carotenoid β-cryptoxanthin since it has excellent bioavailability, and in maize may be nearly as effective as β-carotene in providing retinol to humans. This study was designed to enhance our understanding of the genetic control of: levels of β-cryptoxanthin, conversion of β-carotene into β-cryptoxanthin and zeaxanthin, conversion of β-cryptoxanthin into zeaxanthin, and flux into and within the β-branch of carotenoid pathway. A biparental population derived from two inbreds with relatively high levels of β-cryptoxanthin and different ratios of β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin was studied. Three field replications of this F_2:3 population were grown, grain analyzed by liquid chromatography (LC), and composite interval mapping (CIM) performed to identify 90 quantitative trait loci (QTL) for carotenoids. We detected QTL for β-carotene/(β-cryptoxanthin + zeaxanthin) and (β-carotene + β-cryptoxanthin)/zeaxanthin ratios that contain candidate gene hydroxylase 4 (hyd4), which has not been previously associated with QTL for carotenoids in maize grain. Two color assessment methods, visual score and chromameter reading, were used to phenotype one replicate of the population for initial assessment as simple alternative measuring procedures. A common finding for LC and chromameter analysis included QTL on chromosome 5 that contain candidate gene lycopene β cyclase (lcyβ).     

Alternative pathways of carotene cyclisation in Phycomyces blakesleeanus

Three carotene substrates, lycopene-[¹⁴C], γ-carotene-[¹⁴C] and neurosporene-[¹⁴C] were each incorporated into β-carotene using a cell extract of the C115 car-42 mad-107(?) mutant of P. blakesleeanus. Neurosporene-[¹4C] was also converted into β-zeacarotene and lycopene. Addition of either unlabelled lycopene or β-zeacarotene with neurosporene-[¹⁴C] to the cell extract caused approximately equal reductions of incorporation into β-carotene, indicating that both cyclisation pathways are of equivalent quantitative importance.     

Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and β-cryptoxanthin by ferret carotene-9',10'-monooxygenase

Xanthophyll carotenoids, such as lutein, zeaxanthin and β-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,15′-monooxygenase (CMO1) has been shown to be involved in vitamin A formation, while recent studies suggest that carotene-9′,10′-monooxygenase (CMO2) may have a broader substrate specificity than previously recognized. In this in vitro study, we investigated baculovirus-generated recombinant ferret CMO2 cleavage activity towards the carotenoid substrates zeaxanthin, lutein and β-cryptoxanthin. Utilizing HPLC, LC–MS and GC–MS, we identified both volatile and non-volatile apo-carotenoid products including 3-OH-β-ionone, 3-OH-α-ionone, β-ionone, 3-OH-α-apo-10′-carotenal, 3-OH-β-apo-10′-carotenal, and β-apo-10′-carotenal, indicating cleavage at both the 9,10 and 9′,10′ carbon–carbon double bond. Enzyme kinetic analysis indicated the xanthophylls zeaxanthin and lutein are preferentially cleaved over β-cryptoxanthin, indicating a key role of CMO2 in non-provitamin A carotenoid metabolism. Furthermore, incubation of 3-OH-β-apo-10′-carotenal with CMO2 lysate resulted in the formation of 3-OH-β-ionone. In the presence of NAD⁺, in vitro incubation of 3-OH-β-apo-10′-carotenal with ferret hepatic homogenates formed 3-OH-β-apo-10′-carotenoic acid. Since apo-carotenoids serve as important signaling molecules in a variety of biological processes, enzymatic cleavage of xanthophylls by mammalian CMO2 represents a new avenue of research regarding vertebrate carotenoid metabolism and biological function.     

Carotenoid composition in Zea mays developed at sub-optimal temperature and different light intensities

The content and composition of pigments were examined in the third leaf of Zea mays L. plants grown under controlled environment at near-optimal temperature (24°C) or sub-optimal temperature (14°C) at a light intensity of either 200 or 600 μmol m^?2 s^?1. Compared to leaves grown at 24°C, leaves grown at 14°C showed a large reduction in the chlorophyll (Chl) content, a marked decrease in the Chl a/b ratio, and a large increase in the ratio of total carotenoids/Chl a+b. Leaves grown at 14°C showed a much lower content of β-carotene than leaves grown at 24°C, while the content of the carotenoids of the xanthophyll cycle (violaxanthin [V] + antheraxanthin [A] + zeaxanthin [Z]) was markedly higher in the former leaves as compared to the latter leaves; neoxanthin and lutein were affected by the growth temperature to a much lesser extent. The xanthophylls/β-carotene ratio was about three times higher in leaves grown at 14°C as compared to leaves grown at 24°C. On a chlorophyll basis, the two types of leaves hardly differed in their level of β-carotene, while the levels of the xanthophylls (including lutein and neoxanthin) were higher in 14°C-grown leaves as compared to 24°C-grown leaves. In leaves grown at 14°C, 40 and 56% of the V+A+Z pool was in the form of zeaxanthin at low light intensity and high light intensity, respectively. Only trace amounts of zeaxanthin, if any, were present in leaves grown at 24°C. The changes in the pigment composition induced by growth at sub-optimal temperature were more pronounced at a light intensity of 600 as compared to 200 μmol m^?2 s^?1. In the given range, the light intensity slightly affected the composition of pigments in leaves grown at 24°C. The physiological significance of the modifications to the pigment composition induced by growth at sub-optimal temperature is discussed.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

The biosynthesis,metabolism and translocation of β-amyrin in Sorghum bicolor

The biosynthesis of [¹⁴C] β-amyrin and three other labelled 3β-hydroxypentacyclic triterpenes from [2-^14C] acetate in leaves of Sorghum bicolor was demonstrated. Evidence for the metabolism of [¹⁴C] β-amyrin to the corresponding C-3 ketone (β-amyrone) and for the transport of [¹⁴C] β-amyrin in leaves was also obtained.     

Chlorophyll and carotenoids of five isolates of the red alga Antithamnion plumula

Female plants of five isolates (including two varieties) of the red alga Antithamnion plumula were cultured mnoalgally and analysed for chlorophylls and carotenoids. The relative amounts of the individual pigments ewre similar for the five isolates. All isolates contained chlorophyll β,β-carotene, β,ε-carotene, α-cryptoxanthin and lutein. Allylic methylation of α-cryptoxanthin indicated a 3′-position of the hydroxyl group. An earlier tentative identification of neoxanthin in A. plumula could not be confirmed.     

Ketocarotenoid biosynthesis by Haematococcus lacustris

Haematococcus lacustris incubated on a nutrient-depleted medium utilised acetate-[2-¹⁴C] from the medium and carbon fixed photosynthetically for the biosynthesis of ketocarotenoids. Conversion of β-carotene to astaxanthin occurs via the intermediates echinenone and canthaxanthin.     

Carotenoids in fish. XXVI. Pungitius pungitius (L.) and Gasterosteus aculeatus L. (Gasterosteidae)

The author investigated the presence of various carotenoids in the different parts of the body of Pungitius pungitius (L.) and Gasterosteus aculeatus L. by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

in Pungitius pungitius. α-carotene, β-carotene, β-cryptoxanthin, mutatochrome, zeaxanthin and astaxanthin;

in Gasterosteus aculeatus: β-carotene, β-cryptoxanthin, β-carotene epoxide, neothxanthin, canthaxanthin, mutatochrome, lutein, phoenicoxanthin, zeaxanthin, taraxanthin, tunaxanthin, astaxanthin, astaxanthin ester and α-doradexanthin. The total carotenoid content ranged from 2.229 to 138.504 µg/g wet weight.

     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Enzymatic synthesis of carotenes by cell-free preparations of fruit of several genetic selections of tomatoes

Several mutants of tomatoes are known in which the carotene content of the fruit is markedly altered qualitatively and quantitatively from that found in the standard red tomato variety. These selections are: rr (yellow flesh, low carotene); tt (tangerine, orange, proneurosporene and prolycopene); at at (apricot, low in acyclic carotenes); og^c og^c (crimson, high in lycopene); Verkerk 377-2αα (probably identical to vircscent orange vo vo, high in ζ-carotene); B B (H_i-β, high in β-carotene), and Del Del (Hi-δ, high in δ-carotenc). Studies of carotene synthesis from [1-¹⁴C]isopentenyl pyrophosphate, [¹⁴C]phytoene, and [¹⁴C]lycopcne by soluble enzyme systems obtained from fruits of these selections have shown unexpected enzyme activities. All selections evidence activity for the synthesis of phytoene. All mutants have also been found to contain an enzyme system for the synthesis of β-carotenefrom lycopene. Three of the selections analyzed (rr, at at, and og^c og^c) also contain an enzyme system for the conversion of lycopene to α-carotene and the variants rr and tt contain an enzyme for the synthesis of poly-cis-carotencs from isopentenyl pyrophosphate and phytocne.The reasons for the discrepancies that are observed between carotene composition of fruit of field-grown tomato selections and enzyme activities for carotene synthesis by cell-free preparations obtained from these fruits are not presently known. It is obvious, however, that either inhibitors are present, cofactors are missing, or there are permeability barriers to substrate or cofactor transport into plastids of selections in which enzyme activities are not expressed in field-grown fruit. Further investigations will be required for clarification of this problem.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

Comparative studies of carotenoids in the fauna of the Gullmar Fjord (Bohuslan,Sweden) III. Echinodermata

The author investigated the presence of various carotenoids in the Echinodermata from Gullmar Fjord (Bohuslan, Sweden) by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following:

- inHenricia sanguinolenta:β-carotene, echinenone, canthaxanthin, guraxanthin, lutein-5, 6-epoxide and astaxanthin.

- inAmphiura filiformis: canthaxanthin, cryptoxanthin, lutein, lutein-5, 6-epoxide, isozeaxanthin, zeaxanthin, astaxanthin and 4-hydroxy-4-keto-β-carotene.

- inAmphipholis squamata:β-carotene, cryptoxanthin, lutein, lutein-5, 6-epoxide, astaxanthin, astaxanthin ester, asterin-acid and rubixanthin derivative.

- inOphiopholis aculeata: canthaxanthin, cryptoxanthin, isozeaxanthin, astaxanthin, astaxanthin ester, asterinacid, 4-hydroxy-4-keto-β-carotene, hydroxy rubixanthin and gazaniaxanthin-like substances.

- inOphiothrix fragilis: canthaxanthin, lutein-5, 6-epoxide, isozeaxanthin, astaxanthin, astaxanthin ester, 4-hydroxy-4-keto-β-carotene, and hydroxy rubixanthin.

- inAntedon petatus:canthaxanthin, guaraxanthin, isozeaxan-thin, zeaxanthin, astaxanthin, astaxanthin ester and 4-keto-4-ethoxy-β-carotene.

- inEchinocardium cordatum:β-carotene,γ-carotene, canthaxanthin, lutein, isozeaxanthin, zeaxanthin, astaxanthin and astaxanthin ester.

- inSpatangus purpureus: isozeaxanthin, astaxanthin, astaxanthin ester and 4-hydroxy-4-keto-β-carotene.

     

Molecular diversity, metabolic transformation, and evolution of carotenoid feather pigments in cotingas (Aves: Cotingidae)

Carotenoid pigments were extracted from 29 feather patches from 25 species of cotingas (Cotingidae) representing all lineages of the family with carotenoid plumage coloration. Using high-performance liquid chromatography (HPLC), mass spectrometry, chemical analysis, and ¹H-NMR, 16 different carotenoid molecules were documented in the plumages of the cotinga family. These included common dietary xanthophylls (lutein and zeaxanthin), canary xanthophylls A and B, four well known and broadly distributed avian ketocarotenoids (canthaxanthin, astaxanthin, ??-doradexanthin, and adonixanthin), rhodoxanthin, and seven 4-methoxy-ketocarotenoids. Methoxy-ketocarotenoids were found in 12 species within seven cotinga genera, including a new, previously undescribed molecule isolated from the Andean Cock-of-the-Rock Rupicola peruviana, 3??-hydroxy-3-methoxy-??,??-carotene-4-one, which we name rupicolin. The diversity of cotinga plumage carotenoid pigments is hypothesized to be derived via four metabolic pathways from lutein, zeaxanthin, ??-cryptoxanthin, and ??-carotene. All metabolic transformations within the four pathways can be described by six or seven different enzymatic reactions. Three of these reactions are shared among three precursor pathways and are responsible for eight different metabolically derived carotenoid molecules. The function of cotinga plumage carotenoid diversity was analyzed with reflectance spectrophotometry of plumage patches and a tetrahedral model of avian color visual perception. The evolutionary history of the origin of this diversity is analyzed phylogenetically. The color space analyses document that the evolutionarily derived metabolic modifications of dietary xanthophylls have resulted in the creation of distinctive orange-red and purple visual colors.     

The occurrence and subcellular localization of 3 -hydroxysteroid dehydrogenase in adult rat brain

The subcellular localization of 3α-hydroxysteroid dehydrogenase, the dependency of the rate of reaction on time, concentration of protein, cofactor requirements and the substrate stereospecificity were investigated in the adult rat brain. The $\text{in vitro}$ conversion of 3-keto-5β-cholanoic-24-¹⁴C acid to lithocholic acid was shown to occur in the cytosol without added cofactors. Incubation of ¹⁴C labeled 3α,7α-dihydroxy-5β-cholanoic and 3α,12α-dihydroxy-5β-cholanoic acids with adult rat brain cell-free preparations resulted in the production of less polar metabolites identified as 3-keto-7α-hydroxy-5β-cholanoic and 3-keto-12α-hydroxy-5β-cholanoic acids by TLC, GLC combined with a radioactive monitoring detection system and by cocrystallization to constant specific activity.