IDENTIFICATION AND PHYLOGENETIC IMPLICATIONS OF FATTY ACIDS AND STEROLS IN THREE GENERA OF AQUATIC PHYCOMYCETES

Allomyces macrogynus, A. arbuscula, A. javanicus, Allomyces male and female hybrid strains, Blastocladia ramosa and Monoblepharella sp. were examined for their fatty acid and sterol compositions by GLC and combined GLC/MS. All the organisms produce a range of fatty acids 12 to 20 carbon atoms in length. Palmitic, stearic, and arachidic acid represent the highest concentrations of saturated fatty acids; oleic, linoleic, and arachidonic acid the highest unsaturated fatty acids. B. ramosa synthesizes only two polyunsaturates, linoleic and linolenic, but Allomyces and Monoblepharella are capable of desaturation as far as arachidonic acid. Cholesterol is produced by all the isolates and is the dominant sterol in Allomyces. 24-Methyl and 24-ethyl derivatives of cholesterol are the dominant sterols of Monoblepharella. B. ramosa contains a more complex sterol mixture representing changes which occur in the formation of cholesterol from lanosterol: 24-dihydrolanosterol, 14α-methyl Δ⁸-cholestenol, Δ⁸⁽⁹⁾-cholestenol, 14α-methyl Δ⁷-cholestenol, Δ⁷-cholestenol and cholesterol. Δ⁷-cholestenol, 24-dihydrolanosterol, and 14α-methyl Δ⁸-choIestenol appear to be the major components. This is the first time that 14α-methyl Δ⁸ and 14α-methyl Δ⁷-cholestenol have been reported as naturally occurring sterols.     

Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes.

The stoichiometry of 4-methyl sterol oxidase has been investigated by concurrent assays of rates of oxygen consumption, oxidation of reduced pyridine nucleotide, and formation of steroid 4alpha-oic acid, which is the oxidized product of attack of 4-methyl sterol precursors of cholesterol. The basal, steroid-independent rates of oxidation of alpha-NADH and alpha-NADH-dependent oxygen consumption by rat liver microsomes are about 10 to 15% of the rates observed with beta-NADH. Thus, alpha-NADH is substituted for beta-NADH; alpha-NADH oxidation is observed spectrophotometrically. The slow rate of oxygen consumption is measured accurately with a galvanic oxygen electrode that is attached to an offset amplifier. For maximal velocity, 4alpha-hydroxymethyl-5alpha-cholest-7-en-3beta-ol is the steroid substrate, and oxidase activity is induced 2-fold with a dietary bile acid sequestrant. Under these conditions, accurate measurements are obtained for substrate-dependent increments, which are equal to or greater than basal, substrate-independent rates. For each equivalent of hydroxymethyl group oxidized to carboxylic acid, 2 eq each of oxygen and alpha-NADH are consumed. Thus, the stoichiometry is consistent with that expected for two sequential attacks of the 4alpha-hydroxymethyl group by an external mixed function oxidase. In addition to establishing the stoichiometry of the 4-methyl sterol oxidase, the results further demonstrate that the steroidal 4alpha-carboxylic acid is formed from the hydroxymethyl intermediate by catalysis of a mixed function oxidase rather than dehydrogenases.     

Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to γ-ketotriazole (γ-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of γ-kt showed that their Δ⁵-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ-kt, displayed a sterol content strongly enriched in 14α-methyl sterols and depressed in physiological Δ⁵-sterols. When the transgenic calli were cultured in mixtures of γ-kt and LAN14DM inhibitors sterol compositions enriched in 14α-methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.     

INVESTIGATION OF 4-METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red-tide blooms, were cultured to investigate the 4-methyl sterol constituents. To ascertain a possible influence of cell age on the 4-methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin-layer chromatography. The 4-methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography-mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4-methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24-dimethylcholestan-3-ol as a unique constituent, dinosterol was the major component. Moreover, 4,24-ethylcholestan-3-ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4-methylcholestan-3-ol, 4,24-dimethylcholesta-22-en-3-ol, and 4-methyl-24-ethylcholestan-3-ol. 4-Methyl-24-methylene-cholestan-3-ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4-methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4-methyl-24-ethylcholesta-8(14)-en-3-ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side-chain modifications occur in dinoflagellate 4-methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl-4-methyl sterols under electronic impact is also reported.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

Inhibition of Sterol Biosynthesis in Monilinia fructigena by the Fungicide,S-1358

When Moniiinia fructigena was treated with S–1358 at a concentration of 10 μm, both quality and quantity of digitonin-precipitable sterols were markedly altered. The amount of ergosterol which is a major sterol in the control culture was reduced by S–1358 and the concomitant accumulation of obtusifoliol (one of 4α-methyl sterols) and 24-methylenedihy-drolanosterol (one of 4,4-dimethyl sterols) was observed. The time course study of acetate-U-¹⁴C incorporation into the digitonin-precipitable sterols revealed that 4α-methyl sterols accumulated slowly in the treated culture, while 4,4-dimethyl sterols accumulated rapidly. The accumulation of the sterols containing “extra1” methyl groups suggests that S–1358 blocks demethylation reactions in the conversion from lanosterol to ergosterol in M. fructigena.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra

The complex sterol mixture isolated from $\text{A, nigra}$ was found to contain a low level of Δ⁴-3-keto steroids, 5β-stanols and 4α-methyl sterols in addition to regular (4-demethyl) sterols. The following new marine sterols were isolated and identified using MS and 360 MHz NMR: 5β-cholest-22E-en-3β-ol, 24S-methyl-5β-cholest-22E-en-3β-ol, 24-methylene-5β-cholestan-3β-ol, both epimers at C-24 of 4α-methyl-24-ethyl-5α-cholest-22E-en-3β-ol, 4α, 22ξ, 23ξ-(or 24ξ-)trimethyl-5α-cholest-8(14)-en-3β-ol and (22S, 23S, 24S)-4α-24-dimethyl-22, 23-methylene-5α-cholestan-3β-ol. The latter sterol and 23-demethylgorqosterol have opposite configurations at C-22, C-23, and C-24; the Δ⁸⁽¹⁴⁾ sterol has an unprecedented side chain.     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

Kinetic Analysis of the Three-step Steroid Aromatase Reaction of Human Cytochrome P450 19A1

Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k_cat of 0.06 s⁻¹ for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone.     

Functional expression of Arabidopsis thaliana sterol glycosyltransferase from stably transformed Drosophila melanogaster S2 cells

Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production. Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. Radiometrical assay using uridine diphospho-d-[U-¹⁴C]glucose (UDP-¹⁴C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach ¹⁴C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-sitosterol, which was similar to the recombinant SGT produced by a bacterial expression system. The transfer of ¹⁴C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated ¹⁴C-glucosetransferred β-sitosterol and stigmasterol reactants.     

8-Hydroxyflavonoid glucuronides of <Emphasis Type="Italic">Malope trifida</Emphasis>

The new flavonoid: herbacetin 3-O-β-glucopyranoside-8-O-β-glucuronopyranoside (1) together with known gossypetin 3-O-β-glucopyranoside - 8-O-β-glucuronopyranoside (2) and isoscutellarein: 8-O-β-glucuronopyranoside (3) as well as 4′-methyl ether-8-O-β-glucuronopyranoside (4), were isolated from the calyx and epicalyx leaves of Malope trifida and identified on the basis of their spectroscopic properties: UV, ¹H and ¹³C NMR, ESI/MS. Two other flavonoids were identified as isoscutellarein: 3′-hydroxy 4′-methyl ether-8-O-β-glucuronoside (5) and 8-O- rhamnoglucoside (6) on the basis of their UV and ESI/MS data.     

OCTOPAMINE IN MAMMALIAN BRAIN: RAPID POST MORTEM INCREASE AND EFFECTS OF DRUGS

A modified enzyme radiochemical assay for octopamine, based upon the N-methylation of octopamine by the enzyme phenylethanolamine N-methyl transferase (S-Adenosyl-1-methionine: phenylcthanolamine N-methyl transferase EC 2.1.28), has been developed. [³H]Methyl-S-adenosyl-l- methionine was used as methyl donor, and the reaction products separated by thin-layer chromatography prior to liquid scintillation counting. The method had a sensitivity of about 100 pg, and was suitable for the measurement of endogenous octopamine levels in mammalian brain. Although the method could be used for the determination of phenylethanolamine with similar sensitivity, concentrations of this amine in brain were too low for routine measurement. Octopamine levels in the brains of a number of mammalian species were determined using this procedure. Concentrations of the amine in mouse brain were lower in animals killed by rapid freczing than in animals killed by decapitation; a further increase in brain octopamine took place post-mortem. Brain octopamine was increased following treatment with MAO inhibitors, p-chlorophenylalanine, phenylalanine, tyrosine or phenylethylamine. The effects of tyrosine and phenylethylamine were greatly increased by pretreatment with a monoamine oxidase inhibitor. The antidepressants imipramine and iprindole gave rise to increased brain octopamine concentrations, possibly through an effect upon monoamine oxidase. Administration of chlordiazepoxide chlorpromazine, thyroxine, or reserpine had no effect upon brain octopamine.     

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8(14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ⁸⁽¹⁴⁾ sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ⁸⁽¹⁴⁾ sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Solubilization and partial purification of a microsomal 3-ketosteroid reductase of cholesterol biosynthesis

The rat liver microsomal enzyme that catalyzes NADPH-dependent reduction of 3-ketosteroid intermediates of cholesterol biosynthesis from lanosterol has been solubilized. Although the specific activity has been enhanced only modestly, 24-fold, the solubilized and partially purified reductase can be obtained free of 4-methyl sterol oxidase (also NAD(P)H dependent) and 4α-steroidoic acid decarboxylase (NAD dependent) that are the other two constitutive enzymes of microsomal sterol 4-demethylation. In addition, the isolated protein can be incorporated into artificial phospholipid membranes with retention of activity. Thus, the partially purified 3-ketosteroid reductase is suitable for reconstitution with other enzymes and electron carriers to achieve the 10-step oxidative removal of the 4-gem-dimethyl group of sterols. Both the solubilized and microsomalbound enzyme are essentially inactive with NADH. Also, similar sterol substrate specificities with 4α-monomethyl- and 4,4-dimethyl-3-ketosteroids, pH optima, and other properties of microsomal-bound and solubilized 3-ketoreductase are observed. As observed for other microsomal enzymes the K_m of the solubilized enzyme is significantly lower than that of the membrane-bound enzyme. Membrane-bound 3-ketosteroid reductase is stimulated two- to- threefold by cytosolic Z protein (fatty acid binding protein), but stimulatory activity is lost after solubilization of the microsomal enzyme. Stimulation could not be restored by incorporating the partially purified reductase into an artificial membrane. Stimulation can be reversed by titration of Z-protein with either fatty acids or anti-Z-protein immunoglobulin. Thus, Z protein may modulate several microsomal enzymic activities of sterol biosynthesis in concert by exhibiting affinities for the membrane as well as low-molecular-weight cofactors, substrates, and metabolic effectors.     

A rapid and simple radioassay for inosinic acid: pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa.

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10^?5m allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) into the reaction mixture, the [8-¹⁴C]hypoxanthine (Hx) is converted only to [8-¹⁴C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase.Using [8-¹⁴C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 mm ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8-¹⁴C]IMP or [8-¹⁴C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value.Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.     

THE FATTY ACID AND STEROL COMPOSITION OF FIVE MARINE DINOFLAGELLATES

The fatty acid and sterol compositions of five species of marine dinoflagellates (Scrippsiella sp. Symbiodinium microadriaticum Freud, Gymnodinium sp., Gymnodinium sanguineum Hirasaki, and Fragilidium sp.) are reported. All contained the major fatty acids that are considered common in dinoflagellates, but the proportions were quite variable, and some species contained low contents of some polyunsaturated fatty acids. Concentration ranges for the major fatty acids were: 16:0 (9.0%–24.8%), 18:4(n-3) (2.5%–11.5%), 18:5(n-3) (7.0%–43.1%), 20:5(n-3) (EPA) (1.8%–20.9%), and 22:6(n-3) (DHA) (9.9%– 26.3%). Small amounts of novel very-long-chain highly unsaturated C₂₈ fatty acids occurred in all species. Each dinoflagellate contained a complex mixture of 4-methyl sterols and 4-desmethyl sterols. Four species contained cholesterol, although the amounts were highly variable (from 0.2% of total sterols in Scrippsiella sp. to 45.6% in Fragilidium sp.). All but G. sanguineum contained the 4-methyl sterol dinosterol, and all species contained sterols lacking a double bond in the ring system (i.e. stanols); in Scrippsiella sp. cholestanol composed 24.3% of the total sterols. Other common features of the 4-methylsterol profiles were the presence of 23,24-dimethyl alkylation and unsaturation at Δ²² in the side chain. In Scrippsiella sp., four steroidal ketones were identified: cholestanone, dinosterone, 4α,23,24-trimethyl-5α-cholest-8(14)-en-3-one, and dinostanone. The structures of these corresponded to the major sterols in this species, suggesting that the sterols and steroidal ketones are biosynthetically linked. Steroidal ketones were not detected in the other species. Although fatty acid profiles can be used to distinguish among algal classes, they were not useful for differentiating among dinoflagellate species. In contrast, whereas some taxonomic groupings of dinoflagellates display similar sterol patterns, others, such as the gymnodinoids studied here, clearly do not. The combination of fatty acid, sterol, and steroidal ketone profiles may be useful complementary chemotaxonomic tools for distinguishing morphologically similar species. The identification of steroidal ketones supports earlier suggestions that certain dinoflagellates might be a significant source of such components in marine environments.