Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Quinone methide sclerotization: A revised mechanism for β-sclerotization of insect cuticle

Molecular mechanisms responsible for the stiffening and tanning of insect cuticle are reviewed. Two mechanisms, viz., quinone tanning and β-sclerotization, both involving catecholamine derivatives as sclerotizing precursors, are known to strengthen the cuticle. Quinone tanning mechanism invokes the generation and reactions of o-benzoquinones as the sclerotizing agents, whereas β-sclerotization dictates the activation of catecholamine side chains prior to their incorporation into cuticle. The reactive intermediate for the latter process was proposed by other workers to be 1,2-dehydro-N-acetyldopamine and its quinone. The role of these two compounds in β-sclerotization is critically evaluated. Based on our observation that incubation of cuticular enzyme from Sarcophaga bullata with 4-alkylcatechols results in the production of soluble side chain oxygenated compounds and the formation of catechol-cuticle adducts, an alternate mechanism for β-sclerotization is proposed. This mechanism calls for the generation of quinone methides, tautomers of 4-alkyl-quinones, as the initial products of oxidation of catecholamine derivatives in cuticle. Quinone methides formed spontaneously react with available nucleophiles in cuticle, resulting in the generation of catechol-cuticle adducts and side chain hydroxylated products. Further oxidation of adducts and coupling to other structural units ensure crosslinking of cuticular components. The proposed quinone methide sclerotization accounts for all the chemical observations made on the β-sclerotized cuticle.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

Further studies on the mechanism of oxidation of N-acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2-dehydro-NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2-dehydro-NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β-sclerotization involving quinone methides as the reactive species is presented.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Cuticular sclerotization in the beetles Pachynoda epphipiata and Tenebrio molitor

The sclerotization of cuticle in two species of beetles, Pachynoda epphipiata and Tenebrio molitor, has been investigated and compared with the sclerotization in the locust, Schistocerca gregaria. Two types of sclerotization, β-sclerotization and quinone tanning, occur in all three species. The main type is β-sclerotization, i.e. cross-linking of proteins by means of N-acetyldopamine which is connected to the proteins through the β-position of its side chain. β-Sclerotization is completed in P. epphipiata when it leaves its cocoon, whereas in adult locusts and in adult Tenebrio β-sclerotization continues for several weeks. The cuticle of all three species contains an insoluble enzyme which activates the β-position of N-acetyldopamine and is presumably responsible for the formation of the cross-links. Locust cuticle contains also small amounts of another enzyme which activates the aromatic ring of N-acetyldopamine, resulting in the formation of an o-quinone, which may be involved in quinone tanning of the cuticle. At emergence adult Tenebrio cuticle is rich in both enzymes, but the quinone-forming enzyme is inactivated after a few days, whereas the β-enzyme first decreases and later increases in activity, so that the β-enzyme is the dominating activity in the cuticle of mature adult Tenebrio. The quinone-forming enzyme is presumably responsible for the formation of the brown colour of Tenebrio exocuticle.The exocuticle of adult beetles contains 3,4-dihydroxyphenylacetic acid, which, although it is not easily extracted from the cuticle, is not covalently bound to cuticular components. In Tenebrio it appears in the cuticle a few days after the final ecdysis.The amino acid compositions of both larval, pupal, and adult cuticle from P. epphipiata have been determined, and they are compared with the composition of the cuticle of the corresponding stages of Tenebrio.     

Oxidation of N-β-alanyldopamine by insect cuticles and its role in cuticular sclerotization

The oxidation products formed when various types of insect cuticle were incubated with N-β-alanyldopamine (NBAD) have been studied by means of reversed phase high performance liquid chromatography, and compared to the corresponding products obtained when N-acetyldopamine (NADA) was incubated with the cuticles. The results indicate that NBAD is oxidized to o-quinone and quinone methide derivatives. In contrast, NADA can be oxidized by some cuticles not only to o-quinone and quinone methide derivatives, but it can also be desaturated to α,β-dehydro-N-acetyldopamine, a probable intermediate in β-sclerotization. Some implications for in vivo sclerotization are discussed.     

Nonenzymatic transformations of enzymatically generated N-acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone

We have recently demonstrated that the side chain hydroxylation of N-acetyldopamine and related compounds observed in several insects is caused by a two-enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155-158]. During our studies on o-quinone/p-quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N-acetyldopamine was hydrated to yield N-acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222-6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N-acetyldopamine quinone to iminochrome-type compound(s). The importance of quinone methides in these reactions is discussed.     

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [¹⁴C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of ¹⁸O₂ led to the isolation of 1-naphthol that contained 94% ¹⁸O. The metabolism of [1-²H]-and [2-²H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-²H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The ¹⁸O₂ and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.Abbreviations HPLC High performance liquid chromatography - NMR nuclear magnetic resonance     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.     

Peroxisomal oxidation of lignoceric acid in rat brain

The oxidation of [1-¹⁴C]lignoceric acid was studied in different subcellular fractions of rat brain. The highest specific activity for oxidation of [1-¹⁴C]lignoceric acid to acetate was observed in the light mitochondrial fraction. The oxidation of [1-¹⁴C]lignoceric acid had an absolute requirement for CoASH and ATP. It was stimulated by NAD and FAD by 400 and 280 percent, respectively, whereas addition of carnitine and KCN had no effect. These properties suggest that in brain [1-¹⁴C]lignoceric acid is oxidized in peroxisomes.     

Catabolism of 2-methyloctanoic acid and 3beta-hydroxycholest-5-en-26-oic acid

1. 2-Methyl[1-¹⁴C]octanoic acid was synthesized from 2-bromo-octane and ¹⁴CO₂. 2. 2-Methyl[1-¹⁴C]octanoic acid was readily oxidized to propionic acid and carbon dioxide by mitochondrial preparations from liver, less readily oxidized by adrenal and kidney (mitochondria), and only poorly oxidized by heart, spleen and brown fat (mitochondria). 3. 3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was rapidly oxidized by mammalian-liver mitochondria to propionic acid and carbon dioxide. Caiman-liver and toad-liver mitochondria also oxidized this steroid acid. 4. The oxidation of propionic acid, octanoic acid and palmitic acid by mitochondrial preparations from these various tissues was also studied. 5. Added carnitine did not stimulate 2-methyloctanoic acid oxidation and feebly stimulated 3β-hydroxycholest-5-en-26-oic acid oxidation. 6. The significance of these results is discussed in relation to sterol catabolism in mammals and non-mammalian species.     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Sesquiterpene biosynthesis: The biosynthesis of quadrone and terrecyclic acid

Feeding of [1-¹³C]- and [1,2-¹³C₂]acetates to cultures of Aspergillus terreus gave labeled quadrone (1) and terrecyclic acid (2) which were analyzed by high-field ¹³C NMR. The patterns of enhancements and couplings were used not only in the analysis of the biosynthetic origins of the two metabolites, but also in the assignment of the ¹³C spectra themselves. The latter assignments were confirmed and further extended by extensive analysis by a combination of ¹H COSY, ¹H¹³C heteroCOSY, and difference NOE spectra of quadrone. The biosynthetic pathway was further probed by incorporation of [3,4-¹³C₂]mevalonate, revealing that formation of 1 and 2 involves cleavage of the 3,4-bond of one of the three mevalonte precursors. The results are consistent with the formation of quadrone and terrecyclic acid by cyclization of farnesyl pyrophosphate (8).