Synthesis of 24S and 24R-hydroxy-[24-3H]vitamin D3 and their metabolism in rachitic rats.

An epimeric mixture of 24-hydroxy-[24-₃H]vitamin D₃ was synthesized by the reduction of 24-ketovitamin D₃ by sodium borotritide. The epimeric mixture was converted to the trimethylsilylether derivatives and subjected to high-pressure liquid chromatography using silica gel columns to separate the 24-hydroxy-[24-₃H]vitamin D₃ isomers. The 24R-hydroxy-[24-₃H] vitamin D₃ induced calcification in rachitic rats while the 24S-hydroxy-[24-₃H] vitamin D₃ had little or no such activity. As both isomers of 24-hydroxy-vitamin D₃ are metabolized to 24,25-dihydroxyvitamin D³, it appears that the 24-hydroxyvitamin D₃-25-hydroxylase does not discriminate between the isomers. Only the R-isomer of 24-hydroxyvitamin D₃ is metabolized to 1,24-dihydroxyvitamin D₃, although only trace amounts of this compound were found 2 days after the administration of 24-hydroxyvitamin D₃. The striking difference in the metabolism of the isomers is the high selectivity of the 1-hydroxylase for R-isomer. It is suggested that the high specificity of biological activity for the R-isomer of 24-hydroxyvitamin D₃ is because of the specificity of the 1-hydroxylation of 24,25-dihydroxyvitamin D₃ for the R configuration.     

A genome-derived (gaa.ttc)24 trinucleotide block binds nuclear protein(s) specifically and forms triple helices

The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc)₂₄ tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc)₂₄ block. Competition experiments using simple (gt.ac)_n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa.ttc)_n/protein complex, a binding constant of 9.3×10⁻⁹ mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa)₂₄ strand. OsO₄ and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc)₂₄ block forms different types of intramolecular triple helices: Under superhelical stress, different *H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc)_n tracts.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Evidence for separate intermediates in the biosynthesis of 24α- and 24β-alkylsterols in tracheophytes

When mevalonate-[2-¹⁴C] was incubated with seeds of Pinus pinea, 23% of the label in sterols was found in trans-24-ethylidenecholesterol, 12% in a mixture of 24α- and 24β-methylcholesterol, and 65% in 24α-ethylcholesterol. However, when the radioactive substrate was lanosterol-[24-³H], label appeared only in the 24-ethylidene- (85%) and the epimeric 24-methylsterols (15%). From the ratios of labels in the ethylidene- and methyl-sterols it was possible to show that the tritium in the 24-C₁ -mixture was incorporated only into the 24β-methyl epimer. The labelling patterns are consistent with a pathway to 24β-alkylsterols via Δ²⁵⁽²⁷⁾-sterols bypassing 24-ethylidenesterols and to 24α-alkylsterols via Δ²⁴⁽²⁸⁾-sterols which are isomerized to Δ²⁴⁽²⁵⁾-sterols prior to reduction.     

Breakdown of corticotropin-(1-24) by mouse brain extracts.

ACTH-(1–24) was rapidly degraded to its constituent amino acids upon incubation with soluble mouse brain preparations. Breakdown had a pH optimum near neutrality, a K_m of 1 × 10^?4m, and a V_max of 63 nmol/mg protein/h. Initially a preferential liberation of the amino acids of the N-terminal region of the hormone was observed, followed shortly by a relatively uniform release of amino acids originating throughout the ACTH-(1–24) sequence. Enzymes cleaving internal bonds appear to play a major role in the degradation. The short lag period between hormone disappearance and amino acid formation indicated that few if any large peptides were accumulated during incubation. Pepstatin, chymostatin, antipain, leupeptin, bacitracin, l-1-tosylamide-2-phenylethylchloromethyl ketone, soybean trypsin inhibitor, and diisopropyl-fluorophosphate had little or no effect on amino acid release. Puromycin, bestatin, and EDTA partially inhibited amino acid formation, affecting mainly the residues in the central and carboxyl portions of ACTH-(1–24). N-Ethylmaleimide strongly inhibited the release of all amino acids, indicating extensive involvement of sulfhydryl peptidases in ACTH-(1–24) breakdown.     

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C₂₈- and C₂₉-Δ²⁴⁽²⁸⁾-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ²⁴⁽²⁵⁾ intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-²H₃,28-¹³C)-24-methylenecholesterol, followed by ¹³C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ²⁴⁽²⁵⁾ intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.     

Sexual Dimorphism in Circadian Physiology Is Altered in LXRα Deficient Mice

The mammalian circadian timing system coordinates key molecular, cellular and physiological processes along the 24-h cycle. Accumulating evidence suggests that many clock-controlled processes display a sexual dimorphism. In mammals this is well exemplified by the difference between the male and female circadian patterns of glucocorticoid hormone secretion and clock gene expression. Here we show that the non-circadian nuclear receptor and metabolic sensor Liver X Receptor alpha (LXRα) which is known to regulate glucocorticoid production in mice modulates the sex specific circadian pattern of plasma corticosterone. Lxrα^-/- males display a blunted corticosterone profile while females show higher amplitude as compared to wild type animals. Wild type males are significantly slower than females to resynchronize their locomotor activity rhythm after an 8 h phase advance but this difference is abrogated in Lxrα^-/- males which display a female-like phenotype. We also show that circadian expression patterns of liver 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and Phosphoenolpyruvate carboxykinase (Pepck) differ between sexes and are differentially altered in Lxrα^-/- animals. These changes are associated with a damped profile of plasma glucose oscillation in males but not in females. Sex specific alteration of the insulin and leptin circadian profiles were observed in Lxα^-/- females and could be explained by the change in corticosterone profile. Together this data indicates that LXRα is a determinant of sexually dimorphic circadian patterns of key physiological parameters. The discovery of this unanticipated role for LXRα in circadian physiology underscores the importance of addressing sex differences in chronobiology studies and future LXRα targeted therapies.     

Physical studies on proteins L3 and L24 from the 50 S subunit of the Escherichia coli ribosome.

Proteins L3 and L24, purified by a nondenaturing method from the 50 S ribosomal subunit of Escherichia coli A19, have been characterized. Both proteins were studied under conditions which resemble those used for reconstitution experiments. They were soluble at approximately 2–3 mg/ml and showed little or no aggregation. These proteins have s⁰_20,w values of 2.0 and 1.5 S, and D_20,w values of 7.6 × 10^?7 and 11.0 × 10^?7 cm² s^?1. Partial specific volumes at 20 °C are 0.730 and 0.740 ml g^?1 for the two proteins. The respective molecular weights determined by sedimentation equilibrium are 24,500 and 12,000. The intrinsic viscosity values for the two proteins are 6.0 and 4.0 ml g^?1. From these hydrodynamic parameters an elongated shape for L3 and a globular shape for L24 have been inferred.     

Observations on the biosynthesis of 24-methylcholesterol and 24-ethylcholesterol by Zea mays

Examination of the sterols of Zea mays shoots has established that the 24-ethylcholesterol is predominately the 24α-epimer, sitosterol, but the 24-methylcholesterol is a mixture of the 24α- and 24β-epimers. After incubation of Z. mays shoots with [2-¹⁴C, (4R)4-³H₁]mevalonic acid the sitosterol had a ³H: ¹⁴C atomic ratio of 2.09:5 which is consistent with previous results indicating that a Δ²⁴⁽²⁵⁾ -sterol is implicated in its biosynthesis. By contrast, the 24α- and 24β-methylcholesterol mixture had a higher ³H: ¹⁴C atomic ratio of 2.82:5. This can be explained by the operation of two routes for the elaboration of the 24-methylcholesterol side chain. One may proceed via Δ²⁴⁽²⁵⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the 24α-methylcholesterol with a ³H: ¹⁴C atomic ratio of 2:5. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³- or a Δ²⁵-sterol intermediate to give the 24β1-methylcholesterol with a ³H: ¹⁴C atomic ratio of 3:5. The proportion of these two labelled compounds in the mixture then determines the observed ³H: ¹⁴C atomic ratio (2.82:5). Some evidence for the formation of a Δ²⁵-compound, cyclolaudenol, by Z. mays shoots was provided by incorporation studies employing either [2-¹⁴C]mevalonic acid or [Me-¹⁴C]methionine as the sterol precursor.     

Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease K_Ca and pCa₅₀, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1–40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1–161, cTnI 1–172, and cTnT 236–285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca²⁺ binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca²⁺ binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.     

Solution equilibria of Alizarin Red S with Al(III) and Ni(II)

The solution equilibria of Alizarin Red S (NaH₂- ARS, or 9,10-dihydro-3,4-dihydroxy-9,10-dioxo-2- anthracene sulphonic acid monosodium salt) with Al(III) and Ni(II) were investigated. A general procedure for speciation and the determination of equilibria was developed from work by Coleman. and Gampp. The results of the analyses gave a K_H2 for NaH₂ARS of 10^5.71. For the AI(III)/Na₂- HARS equilibrium it was determined that a 1:2 complex was formed in solution with β₂ = 10^12.88. For the Ni(II)/NaH₂ARS equilibrium two species exist, a 1:1 and a 1:2 complex with β₁ = 10^5.35 and β₂ = 10^11.53.     

Biosynthesis of cholest-5-ene-3beta, 24-diol (cerebrosterol) by bovine cerebral cortical microsomes

The presence of cholest-5-ene-3β, 24-diol (cerebrosterol) in samples of human, bovine, and rabbit brains has been established by isolation of the sterol therefrom and identification by comparison of physical properties. Cholest-5-ene-3β, 24-diol was present at the level of 66.5 sg/g of dried tissue in human brain, 42.9 μg/g in cattle brain, and 89.5 pg/g in rabbit brain. Cholest-5-ene-3β, 24-diol was the only readily detectable hydroxycholesterol derivative in these brain tissues and was concentrated in the 105,000 g pellet (microsomal fraction) of both human and bovine cerebral cortex, with no demonstrable amounts of the sterol present in nuclear or mitochondrial fractions. Incubation of [1,2-³H]- or of [4-¹⁴C]-cholesterol with the 105,000 g microsomal pellet from bovine cerebral cortical homogenates demonstrated 0.1-0.38 per cent conversions to radioactive cholest-5-ene-3β, 24-diol, isolated and purified as the 3β, 24-dibenzoate. The bioconversion required oxygen, and a stimulation of hydroxylation by added NADPH₂ was demonstrated. Our observations establish that a sterol 24-hydroxylase system is present in bovine cerebral cortex.     

9-Isoleucine]ACTH1-24, a competitive antagonist of ACTH1-24 induced cyclic AMP and corticosterone production.

Substitution of tryptophan⁹ in ACTH_1–24 by isoleucine results in complete loss of biological activity. A dose of 3.4 × 10^?5 M per ml fails to stimulate corticosterone and cyclic AMP production. This analogue inhibits cyclic AMP production and corticosterone production induced by ACTH_1–24 in isolated adrenal cortex cells. The I₅₀ values for corticosterone and cyclic AMP inhibition are 2.3 × 10^?6 M and 3.4 × 10^?6 M respectively.     

Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease K_Ca and pCa₅₀, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1–40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1–161, cTnI 1–172, and cTnT 236–285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca²⁺ binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca²⁺ binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Laser Photofootprinting of d(C)·d(G)·d(G) Intramolecular Triplex

Abstract

Supercoiling-induced structural transition of the d(C₂₄GC₂₁,) · d(G₂₁CG₂₄) sequence in plasmid DNA in the presence of Mg²⁺ at neutral pH results in alterations of efficiencies of not only single-quantum (pyrimidine[6–4]pyrimidone adducts) but also two-quantum (alkalisensitive lesions of dG residues) photomodifications of nucleoside residues within this sequence. The generation of both types of photoreactions was achieved by the application of high-intensity laser UV radiation (intensity ～ 10¹¹ W/m², pulse duration ～ 10^?8 s, λ= 266 nm) for irradiation of a plasmid DNA The modification extent sufficient for analysis of photoreaction efficiency distributions along both strands of the insert (photofootprinting) was obtained by the action of a single nanosecond pulse of laser UV radiation. The pattern of a laser photofootprinting is consistent with the d(C) · d(G) · d(G) triplex formation in the presence of Mg²⁺ within the insert and shows some details of this triplex structure.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Cloning,mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a K_mapp of 38 μM and k_catapp of 0.14 min⁻¹. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-¹³C]- or [24-²H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of ¹H and ¹³CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ²⁴⁽²⁵⁾-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K_i 14 nM) and 26,27-dehydrolanosterol (K_i 54 μM and k_inact of 0.24 min⁻¹) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC₅₀, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C₁-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.     

The conversion of 24-ethylidene sterols into poriferasterol by the alga Ochromonas malhamensis

A convenient method is described for the preparation of fucosterol-[7-³H₂] and 28-isofucosterol-[7-³H₂]. Both of these 24-ethylidene sterols, as well as 5α-stigmasta-7,Z-24(28)-diene-3β-ol-[2,4-³H₄], were converted into the 24β-ethyl sterol, poriferasterol, by cultures of the chrysophyte alga Ochromonas malhamensis. However, fucosterol-[7-³H₂] was not so efficiently incorporated as the other two compounds thus indicating that the configuration of the 24-ethylidene group is of some importance. It is suggested that a 24-ethylidene sterol of the Z-configuration is produced in de novo poriferasterol synthesis and that a Δ^22,24(28)-diene may be an important subsequent intermediate.