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We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.

Total membranes or microsomal fractions prepared from the amnion-derived WISH cell line posses a coenzyme A-independent transacylase activity. The transacylase utilizes 1-alkenyl- and 1-alkyl-2-lysoglycerophospholipids as preferred acceptors. Marginal transacylation was observed with 1-acyl-2-lysoglycerophospholipids. The reaction occurred in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and was not affected by phospholipase A2 inhibitors. Both 1-acyl- or 1-alkyl-glycerophosphocholines containing an arachidonoyl residue in the sn-2 position were effective as donors, while 2-oleoyl- or 2-palmitoyl-glycerophosphocholines were ineffective. The presence of the transacylase, the specificity of the substrates, and the stability of the 1-alkenyl bond provide a biochemical model that may explain the increased proportion of a highly enriched arachidonate-containing phosphatidyl-ethanolamine-plasmalogens fraction that is found in amnion at term.

The final step in the synthesis of n-hydrocarbons in an animal and a higher plant involves enzymatic decarbonylation of aldehydes to the corresponding alkanes by loss of the carbonyl carbon. Whether such a novel reaction is involved in hydrocarbon synthesis in the colonial microalga, Botryococcus braunii, which is known to produce unusually high levels (up to 32% of dry weight) of n-C27, C29, and C31 alka-dienes and -trienes, was investigated. Dithioerythritol severely inhibited the incorporation of [1-14C]acetate into these hydrocarbons with accumulation of the label in the aldehyde fraction in the B. braunii cells. Microsomal preparations of the alga synthesized alkane from fatty acid and aldehyde in the absence of O2. Conversion of fatty acid to alkane required CoA, ATP, and NADH, whereas conversion of aldehyde to alkane did not require the addition of cofactors. That the alkane synthesis involves a decarbonylation was shown by the production of CO and heptadecane from octadecanal. CO was identified by adsorption to RhCl[(C6H6)3P]3. The decarbonylase had a pH optimum at 7.0, an apparent Km of 65 microM, a Vmax of 1.36 nmol/min/mg and was inhibited by the metal chelators EDTA, O-phenanthroline and 8-hydroxyquinoline. It was stimulated nearly threefold by 2 mM ascorbate and inhibited by the presence of O2. A partial (28%) retention of the aldehydic hydrogen of [1-3H]octadecanal in the heptadecane was observed; the remaining 3H was lost to H2O. The microsomal preparation also catalyzed the oxidation of 14CO to 14CO2, with a pH optimum of 7.0. This accounts for the nonstoichiometry of CO to heptadecane observed. In vivo studies with 14CO showed that the label was incorporated into metabolic products. This metabolic conversion of CO, not found in the previously examined hydrocarbon synthesizing systems, may be necessary for organisms that produce large amounts of hydrocarbons such as the present alga. The mechanism of the decarbonylation and the nature of the decarbonylase remain to be elucidated.

The propionyl-CoA condensing enzyme which catalyzes the first step in the biosynthesis of 2-methylbutyrate and 2-methylvalerate by Ascaris muscle appears to exist in at least three forms in the mitochondria of this parasitic nematode. Two forms, A and B, were separated by ion exchange chromatography on CM-Sephadex. Chromatography on phosphocellulose resulted in the recovery of one minor peak (I) and two major peaks with activity (II and III). A and B as well as I, II, and III differed in their specific activities. Forms B and III were the most retained by their resins, and were the most active forms of the enzyme in each case. Inhibition studies with metabolites from Ascaris mitochondria indicate that CoASH, a product of the condensation reaction, and acetyl-CoA are effective inhibitors of the condensing and thiolytic activities of the Ascaris enzyme, respectively. Incubation of the active enzyme form B for 2 h with 0.1 mM CoASH at room temperature under nitrogen caused the loss of 92% of the propionyl-CoA condensing activity and 67% of the thiolase activity when assayed in standard mixtures. The propionyl-CoA condensing enzyme exhibited a hyperbolic dependence of the condensation velocity to changes in substrate concentration. However, in the presence of CoASH the Michaelis-Menten kinetics was transformed into a sigmoidal kinetics indicating a deviation from a simple product inhibition. Inactivation of the most active forms of the enzyme with CoASH was accompanied by (a) a change in the chemical reactivity of the protein toward p-chloromurcuribenzoate, (b) a change in the uv-visible spectrum of the protein, and (c) a change in the elution patterns from both CM-Sephadex and phosphocellulose column chromatography, where-upon one, two, or more protein peaks were obtained. The several protein peaks resolved by rechromatography of the [14C]CoASH-inactivated enzyme III on phosphocellulose had different CoASH contents. The elution positions were correlated with the less active forms (I and II) having increased [14C]CoASH activities. Similarly, the two peaks isolated upon rechromatography of the CoASH-partially inactivated enzyme B on CM-Sephadex had different isotope contents and the elution position of enzyme A corresponded to the less active form. The results described indicate that the CoASH modification of Ascaris propionyl-CoA condensing enzyme may be responsible for the existence of several forms of the enzyme and might represent a mode of control by chemically modulating the amount of the active forms of the enzyme.

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 microM N1,N11-bis(ethyl)norspermine (BENSPM) for 48-72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures--binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.

O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation.

Oxoglutarate oxidation by purified potato mitochondria which had been stored at low temperature for 48 h or longer was stimulated by added coenzyme A. Exogenous coenzyme A was accumulated by potato mitochondria, both freshly prepared and aged, in a manner sensitive to uncouplers and low temperature. Coenzyme A was concentrated approximately 10-fold in the matrix under steady-state conditions. This coenzyme A uptake followed saturation kinetics with an apparent Km of 0.2 mM and a V of 4-6.5 nmol min-1 mg-1 protein, suggesting carrier-mediated transport. This transport was insensitive to an inhibitor of NAD+ transport. It is suggested that plant mitochondria possess a specific carrier for the net accumulation of coenzyme A.

Phosphorylation and inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase in the presence of ATP and Mg2+ requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. Analogs of coenzyme A were also effective in stimulating the inactivation of carboxylase. The KA of CoA for stimulated carboxylase inactivation was 25 microM. The presence of coenzyme A did not alter the Km of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The KD of CoA binding to carboxylase is 27 microM. These results indicate that coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase.

Activation of the NAD malic enzyme is shown to be caused by free, uncomplexed fumarate2-. Mg-fumarate has no detectable effect on the enzyme. Fumarate2- isotherms are biphasic in that they consist of an activating as well as a deactivating region. Activation is shown to result from an increase in the affinity of the enzyme for malate2- while deactivation results from a reduction in Vmax. Phosphate does not affect the response of the enzyme to fumarate2-, while Cl- inhibits the enzyme in a manner that cannot be overcome by fumarate2-. SO42-, another activator of the malic enzyme, reduces the Ka for fumarate2- from 3.9 to 2.1 mM. Activation of the enzyme by coenzyme A (CoA) is hyperbolic with a Ka for CoA of 2.1 microM. Fumarate2- reduces this value to 1.2 microM. CoA, like SO42-, is able to increase the affinity of the enzyme for fumarate2-, decreasing its Ka by 56%. An additional effect of fumarate2- is to cause the interconversion of different catalytic forms of the enzyme which exist when Mg2- is limiting. On the basis of these results, a model of the number and types of allosteric sites present on the NAD malic enzyme is proposed.

Methoxycarbonyl-CoA disulfide has been used as an active-site-directed inhibitor of carnitine acetyltransferase. Stoichiometric addition of methoxycarbonyl-CoA disulfide to carnitine acetyltransferase showed the modification of one sulfhydryl group with concomitant loss of about 80% enzyme activity. The rate of modification of this sulfhydryl group is an order of magnitude faster than that of the remaining sulfhydryl groups in the enzyme. Methoxycarbonyl-CoA disulfide inactivation is biphasic: k1 = 1.09 X 10(2) M-1 S-1, k2 = 1.1 X 10(1) M-1 S-1. This modification, Enz-SS-CoA is covalent; it can be reversed with either dithioerythritol or thiocholine. Acetyl-carnitine and acetyl-CoA protected the enzyme against methoxycarbonyl-CoA disulfide inactivation; however, carnitine did not. These results indicate the presence of a sulfhydryl group in carnitine acetyltransferase at the site of acetyl group transfer. Titration of carnitine acetyltransferase with nonspecific sulfhydryl reagents, DTNB, and rho-nitrophenoxycarbonyl methyl disulfide, revealed that four sulfhydryl groups were preferentially modified by these reagents. The results also show that seven other sulfhydryl groups are available for modification.

Regulation of oxidation of [1-14C]palmitate in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin prepared by use of a Ficoll/sucrose density gradient. The mitochondrial preparation contained considerable Mg2+-ATPase activity, but was virtually free of contamination with nonmitochondrial fractions. Palmitate oxidation was inhibited by increasing the concentration of ATP in the assay system to near-physiological levels (2 mM), and the inhibition at 2 or 4 mM ATP was analyzed by comparing it with palmitate oxidation at near-maximal rates with low levels of ATP (0.5 or 1 mM). Inhibition was increased by the addition of ADP or by increasing the concentration of Mg2+ in the assay system, whereas inhibition was decreased by decreasing the concentration of mitochondrial protein or L-carnitine in the assay system. Increasing CoA concentration also had a deinhibitory effect. With 0.5 or 1 mM ATP, however, neither inhibition by added ADP nor protein concentration-dependent inhibition was observed, and the rate of oxidation was saturated with increasing concentrations of Mg2+, L-carnitine, or CoA. These results indicated that ADP was involved in the inhibition of high rates of palmitate oxidation in the presence of sufficient ATP and L-carnitine. The inhibitory effect of increasing the concentration of mitochondrial protein could be explained by the enhanced amounts of ADP present in the preparation; similarly, increased concentrations of Mg2+ would provide higher levels of ADP by stimulating the Mg2+-ATPase reaction. We discuss the possibility that the transport of ADP across the inner membrane of brain mitochondria is coupled to the inhibition of palmitate oxidation.

Diesters of 3-hydroxy C8, C10, and C12 acids, the female mallard duck pheromones, were found as the major products of the uropygial glands only during the breeding season. The 3-hydroxy acids were identified by mass spectrometry of the trimethylsilyl ethers of the methyl esters and of the diols derived from LiAlH4 reduction of the hydroxy acids. A cell-free extract from the gland catalyzed conversion of dodecanoic acid to 3-hydroxydodecanoic acid which was identified by radio thin-layer and radio gas chromatographic analysis of the enzymic products as methyl-3-acetoxydodecanoate and as diacetate of the diol generated by LiAlH4 reduction of the enzymic product. The enzymic introduction of the hydroxyl group at C-3 was catalyzed mainly by a 50,000g pellet prepared from a 1000g supernatant obtained from the cell-free extract. This reaction required ATP, CoA, and O2, and the CoA ester of the acid was more efficiently converted than the free acid to the 3-hydroxy acid. KCN at 1 mM and 50% CO did not inhibit the reaction. 3H from 3H2O was incorporated into 3-hydroxydodecanoic acid during the enzymic synthesis of this acid from dodecanoic acid. Mass spectrometry of the 3-hydroxy acid generated by the particulate fraction in the presence of H2 18O showed that 18O was incorporated as expected from hydration of a delta 2 double bond. From the above results it is tentatively concluded that peroxisomal acyl-CoA oxidase converts the acyl-CoA to the 2-enoyl-CoA which is hydrated to generate the 3-hydroxy acid.