Depigmenting effect of <Emphasis Type="Italic">Sterculia lynchnophera</Emphasis> on B16F10 melanoma and C57BL/6 melan-a cells

To develop a novel skin depigmenting agent from natural sources, the inhibition of melanogenesis by the Chinese herb, Sterculia lynchnophera (SL), was evaluated. Treatment of B16F10 melanoma cells and melan-a cells with SL exhibited a 32.9% and 68.2% inhibition of melanin synthesis without cytotoxicity at a concentration of 200 μg/ml, respectively. This herb possessed a high free radical scavenging activity with IC₅₀=11.02 μM. The methanol extract of SL slightly inhibited in vitro mushroom tyrosinase activity (23.4% at a concentration of 200 μg/ml) and had a significant inhibitory effect on cellular tyrosinase activivity (48.65% and 88.56% inhibition at the concentration 200 μg/ml in B16F10 cells and C57BL/6 melan-a cells, respectively). From the western blotting results, SL inhibited the expression of tyrosinase and tyrosinase related protein 1 (TRP-1). Taken together, we suggest that SL may be a safe and effective depigmentation agent.     

Depigmenting effect of <Emphasis Type="Italic">Cinnamomum cassia</Emphasis> Presl in B16F10 melanoma cells

To find efficient depigmenting agents, we examined several Chinese herbs for melanogenesis inhibition and toxicity. Cinnamomum cassia Presl exhibited low cytotoxicity at even high concentration (200 μg/ml). The effects on melanogenesis of cultured B16 melanoma cells, mushroom tyrosinase activity, and free radical scavenging activity were further assessed. The methanol extracts of this plant showed the suppression of melanin synthesis. Melanin content was dose-dependently decreased by this herb extract as compared with control cells. It also showed good anti-oxidative activity (IC₅₀=3.7 μg/ml) but no inhibition of mushroom tyrosinase activity. This result showed that Cinnamomum cassia Presl extract might be useful and safe as a new whitening agent in cosmetics.     

Inhibition of melanogenesis by Erigeron canadensis via down-regulating melanogenic enzymes in B16F10 melanoma cells

The effects of Erigeron canadensis extract on melanogenesis and cell toxicity in cultured B16F10 mouse melanoma cells were investigated. E. canadensis extract down regulated melanin synthesis effectively at a non-toxic concentration. Its extract was fractionated by using a recycling HPLC with GS310 column (21.5×500 mm, 10–15 μM) into five fractions. The fraction 1 showed melanin inhibition by 48.0% at 100 mg/ml which was 2.5 times more efficient than the depigmenting effect of commercial arbutin (17.5%) and also did not show cell toxicity. To elucidate the depigmenting mechanism of fraction 1, in vitro and cellular tyrosinase activity, antioxidant activity, and protein level of the main melanogenic enzymes, such as tyrosinase, TRP-1 and TRP-2 were evaluated. Fraction 1 inhibited melanin synthesis in B16F10 melanoma cells by decreasing protein levels of melanogenic enzymes, especially tyrosinase. In conclusion, we suggest that this fraction may be a safe and effective depigmentation agent.     

Potential application of acetone extract of <Emphasis Type="Italic">Astragalus sinicus</Emphasis> Linne seed to functional cosmetics

For the functional cosmetic agent using acetone extract of A. sinicus Linne seed, the effects of whitening, wrinkling, and safety were investigated. Cell viabilities of Raw 264.7 up to 60 mg/mL did not appear to have any significant direct cytotoxic effect. The melanin concentration was decreased up to 62.1% at 20 mg/mL. When the acetone extract concentration of A. sinicus Linne seed was increased from 5 to 20 mg/mL, the inhibitory activity of tyrosinase was sharply increased from 61.3 to 93.8%. However, above 30 mg/mL, it did not increase. The inhibition effects of elastase and collagenase were increased with the extract concentration. Especially, when acetone extract concentration of A. sinicus Linne seed was increased from 25 to 200 μg/mL, the inhibition effect of elastase was increased from 60.2 to 97.5%. The inhibition effect of collagenase was increased from 35.0 to 99.0% when increased from 50 to 300 μg/mL. The indexes of pigment and coarseness were 28.56 MI and 18.45R-value, respectively, after 8 weeks of clinical trial using cream pack containing 0.2% of acetone extract of A. sinicus Linne seed. The indexes of elasticity and moisture were 64.5Ur/Uf and 55.2AU, respectively, after 8 weeks of clinical trial. These results demonstrate that acetone extract of A. sinicus Linne seed may be useful as a potential agent for functional cosmetics.     

Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (<Emphasis Type="Italic">Glycine tomentella</Emphasis>)

I-Tiao-Gung, Glycine tomentella, has been used extensively as a traditional herbal medicine to relieve physical pain, but its bioactivity has not been studied systematically. Ninety-five percent ethanol extracts of G. tomentella (GT-E) showed antioxidant activity in human plasma by prolonging the lag phase (+T_lag) of Cu²⁺-induced, LDL oxidation and were dose dependent. The+T_lag of LDL combined with 3.2 μg/mL GT-E was similar to that with 2.0 μM (ca. 0.5 μg/mL) Trolox. A similar inhibitory effect was found toward tilapia plasma LDL. In addition, GT-E inhibited tilapia thrombocyte (nucleated platelet), 5-, 12-, and 15-lipoxygenase (LOX). The IC₅₀ values were 0.43, 0.72, and 0.42 μg/mL, respectively, whereas the IC₅₀ values for nordihydroguaiaretic acid (NDGA) on 5-, 12-, and 15-LOX were 2.3, 1.6, and 1.7 μg/mL, respectively. The IC₅₀ value for cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL, whereas the IC₅₀ value by indomethacin as a positive control was 0.61 μg/mL. The prevention of LDL oxidation and the dual inhibition of LOX and COX-2 are indicative of the possible roles of I-Tiao-Gung in antiatherosclerosis and anti-inflammation.     

Repositioning of Thiourea-Containing Drugs as Tyrosinase Inhibitors

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC₅₀ of 14 and 15 μM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 μM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.     

Inhibition of lipoxygenase 1 by phosphatidylcholine micelles-bound curcumin

Curcumin (diferuloyl methane) from rhizomes of Curcuma longa L. binds to phosphatidylcholine (PC) micelles. The binding of curcumin with PC micelles was followed by fluorescence measurements. Curcumin emits at 490 nm with an excitation wavelength of 451 nm after binding to PC-mixed micelles stabilized with deoxycholate. Curcumin in aqueous solution does not inhibit dioxygenation of fatty acids by Lipoxygenase 1 (LOX1). But, when bound to PC micelles, it inhibits the oxidation of fatty acids. The present study has shown that 8.6 μM of curcumin bound to the PC micelles is required for 50% inhibition of linoleic acid peroxidation. Lineweaver-Burk plot analysis has indicated that curcumin is a competitive inhibitor of LOX1 with K _l of 1.7 μM for linoleic and 4.3 μM for arachidonic acids, respectively. Based on spectroscopic measurements, we conclude that the inhibition of LOX1 activity by curcumin can be due to binding to active center iron and curcumin after binding to the PC micelles acts as an inhibitor of LOX1.     

Synergistic Effect of DIBOA and Verbascoside from Acanthus mollis Leaf on Tyrosinase Inhibition

Overexpression of melanin contributes to darkening of plant and fruit tissues and skin hyperpigmentation, leading to melasma or age spots. Although melanin biosynthesis is complex and involves several steps, a single enzyme known as tyrosinase is key to regulating this process. The melanogenesis pathway is initiated by oxidation of the starting material l-tyrosine (or l-DOPA) to dopaquinone by tyrosinase; the resulting quinone then serves as a substrate for subsequent steps that eventually lead to production of melanin. Medicinal plants are considered a good source of tyrosinase inhibitors. This study investigated the tyrosinase inhibitory activity of A. mollis leaf extracts and their phytochemicals. Significant activity was verified in the ethanol extract –EEt (IC₅₀ = 1.21 µg/mL). Additionally, a kinetic study showed that this tyrosinase inhibition occurs by DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and verbascoside contribution through a non-competitive reaction mechanism. A synergistic effect on tyrosinase inhibition was observed in the binary combination of the compounds. In conclusion, both EEt and a mixture of two of its phytochemicals can be effective tyrosinase inhibitors and can be used as a bleaching agent for cosmetic formulations in the future.     

Synthesis and phospholipase C inhibitory activity of D609 diastereomers

The potassium xanthate D609 is widely accepted as a selective inhibitor of PC-specific phospholipase C (PC-PLC). The tricyclo[5.2.1.0^2,6]decane skeleton present in D609 can lead to four diastereomeric paris, but the diastereoselectivity of PC-PLC inhibition has never been reported. In this article, the synthesis of racemic D609 diastereomers and that of other xanthates, as well as their inhibitory effect on PC-PLC is reported. All xanthates obtained were competitive inhibitors of PC-PLC from Bacillus cereus (PLCBc). No significant differences were found in the activity of D609 diastereomers (K _i 13–17 μM), suggesting the absence of a diastereochemical control of the enzyme by xanthate inhibitors. This result was confirmed after obtaining other potassium xanthates differing from D609 in the aliphatic chain. Among them, the potassium O-n-decenylxanthate was the most active inhibitor of PLCBc (K _i 10 μM). These data indicate that the essential structural requirements for PLCBc in vitro inhibition by xanthates are the presence of a Zn-chelating dithiocarbonate head and a sufficiently hydrophobic aliphatic moiety.     

<Emphasis Type="Italic">Bidens pilosa</Emphasis> L. Exhibits High Sensitivity to Coumarin in Comparison with Three Other Weed Species

Nine natural plant compounds were screened for phytotoxicity to Bidens pilosa L. a troublesome weed in field and plantation crops. The sensitivity of three other weed species to coumarin, the most active identified compound, was also evaluated. Coumarin, at a concentration of 500 μM, had little effect on germination and growth of Senna obtusifolia L., Euphorbia heterophylla L., and Ipomoea grandifolia L. when compared with its effects on B. pilosa L. In a concentration range of 10–100 μM, coumarin caused a dose-dependent inhibition of germination and growth of B. pilosa L. The measurements of some parameters of energy metabolism revealed that coumarin-treated root tissues exhibited characteristics of seedlings in an earlier stage of growth, including higher respiratory activity and higher activities of alcohol dehydrogenase and lipoxygenase. These results suggest that coumarin inhibition of germination and growth of B. pilosa L. was not a consequence of an impairment of energy metabolism. Rather, it seems to act as a cytostatic agent, retarding germination. At concentrations above 50 μM, coumarin increased lipoxygenase activity and the level of conjugated dienes of root extracts, suggesting that it may induce oxidative stress in seedling roots.     

<Emphasis Type="Italic">9-trans,11-trans</Emphasis>-CLA: Antiproliferative and proapoptotic effects on bovine endothelial cells

Endothelial cell function can be influenced by nutrition, especially dietary FA and antioxidants. One class of dietary FA that is found in meat and dairy products derived from ruminant animals is conjugated linoleic acids (CLA). We have examined the effects of several CLA isomers on endothelial cell proliferation. 9t,11t-CLA was the only isomer that inhibited bovine arotic endothelial cell (BAEC) [³H]methylthymidine incorporation (I₅₀=35 μM), and this antiproliferative effect was time-dependent. A small decrease (20%) in cell number was observed only at the highest concentration (60 μM) tested. The 9c,11t-, 9c,11c-, 10t 12c-, and 11c,13t-CLA isomers did not exhibit any antiproliferative effects over a 5–60 μM concentration range. α-Tocopherol and BHT decreased BAEC proliferation, but pretreatment of cells with either of these antioxidants substantially attenuated the antiproliferative effect of 9t,11t-CLA. No difference in lipid peroxidation, as measured by the thiobarbituric acid assay for malondialdehyde, was observed on treatment of endothelial cells with either 9t,11t- or 9c,11t-CLA. However, a 43% increase in caspase-3 activity was observed after incubating BAEC with 9t,11t-CLA, suggesting that the antiproliferative effect of this isomer is partially due to an apoptotic pathway. In contrast to the above results with normal endothelial cells, these five CLA isomers all inhibited proliferation of the human leukemic cell line THP-1, with the 9t,11t isomer again being the most (I₅₀=60 μM) effective. These results confirm that different CLA isomers have different inhibitory potencies on the proliferation of normal and leukemic cells.     

Regulation of Cell Division and Growth in Roots of <Emphasis Type="Italic">Lactuca sativa</Emphasis> L. Seedlings by the <Emphasis Type="Italic">Ent</Emphasis>-Kaurene Diterpenoid Rabdosin B

Rabdosin B, an ent-kaurene diterpenoid purified from the air-dried aerial parts of Isodon japonica (Burm.f) Hara var. galaucocalyx (maxin) Hara, showed a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in lettuce seedlings (Lactuca sativa L.). Lower concentrations of rabdosin B (20–80 μM) significantly promoted root growth, but its higher levels at 120–200 μM, by contrast, had inhibitory effects. Additionally, all tested concentrations (10–40 μM) inhibited root hair development of seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of rabdosin B at the lower concentrations resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in seedlings’ root tips. In contrast, rabdosin B at higher concentrations inhibited root growth by affecting both cell length in the mature region and division of meristematic cells. Comet assay and cell cycle analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced cell cycle retardation of the G₂ phase and S phase at different times.     

Fumigant and Contact Toxicities of Monoterpenes to <Emphasis Type="Italic">Sitophilus oryzae</Emphasis> (L.) and <Emphasis Type="Italic">Tribolium castaneum</Emphasis> (Herbst) and their Inhibitory Effects on Acetylcholinesterase Activity

A comparative study was conducted to assess the contact and fumigant toxicities of eleven monoterpenes on two important stored products insects—, Sitophilus oryzae, the rice weevil, and Tribolium castaneum, the rust red flour beetle. The monoterpenes included: camphene, (+)-camphor, (−)-carvone, 1-8-cineole, cuminaldehyde, (l)-fenchone, geraniol, (−)-limonene, (−)-linalool, (−)-menthol, and myrcene. The inhibitory effect of these compounds on acetylcholinesterase (AChE) activity also was examined to explore their possible mode(s) of toxic action. Although most of the compounds were toxic to S. oryzae and T. castaneum, their toxicity varied with insect species and with the bioassay test. In contact toxicity assays, (−)-carvone, geraniol, and cuminaldehyde showed the highest toxicity against S. oryzae with LC₅₀ values of 28.17, 28.76, and 42.08 μg/cm², respectively. (−)-Carvone (LC₅₀ = 19.80 μg/cm²) was the most effective compound against T. castaneum, followed by cuminaldehyde (LC₅₀ = 32.59 μg/cm²). In contrast, camphene, (+)-camphor, 1-8-cineole, and myrcene had weak activity against both insects (i.e., LC₅₀ values above 500 μg/cm²). In fumigant toxicity assays, 1-8-cineole was the most effective against S. oryzae and T. castaneum (LC₅₀ = 14.19 and 17.16 mg/l, respectively). Structure-toxicity investigations revealed that (−)-carvone—, a ketone—, had the highest contact toxicity against the both insects. 1-8-Cineole—, an ether—, was the most potent fumigant against both insects. In vitro inhibition studies of AChE from adults of S. oryzae showed that cuminaldehyde most effectively inhibited enzyme activity at the two tested concentrations (0.01 and 0.05 M) followed by 1-8-cineole, (−)-limonene, and (l)-fenchone. 1-8-Cineole was the most potent inhibitor of AChE activity from T. castaneum larvae followed by (−)-carvone and (−)-limonene. The results of the present study indicate that (−)-carvone, 1,8-cineole, cuminaldehyde, (l)-fenchone, and (−)-limonene could be effective biocontrol agents against S. oryzae and T. castaneum.     

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 μM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 μM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(Z) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 μM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11(E) CLA isomer being the most effective and the 9(Z),11(Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The K _i values obtained by Dixon replots range from 18.7 μM for the most effective isomer, 9(Z),11(Z) CLA, to 105.3 μM for the least effective isomer, 9(Z),11(Z) CLA. The K _i value for flurbiprofen with ram seminal vesicle microsomes was 0.33 μM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.     

<Emphasis Type="Italic">Nelumbo nucifera</Emphasis> extracts as whitening and anti-wrinkle cosmetic agent

Water extract from Nelumbo nucifera was tested for possible functional cosmetic agent. Whitening effect was measured by tyrosinase inhibition assay and DOPA-oxidase inhibition assay, and anti-wrinkle effect was checked by elastase inhibition assay. DOPA-oxidase inhibition effect (whitening effect) of Nelumbo nucifera’s leaf, seed and flower extract was 59%, 57% and 50%, respectively. Nelumbo nucifera’s leaf, seed and flower extract showed 56%, 49%, and 54% elastase inhibition (anti-wrinkle effect) at 200 μg/ml, while adenosine indicated 26% inhibition. Water cream including Nelumbo nucifera’s root, leaf, flower, stem extract did not cause significant skin irritation. Water cream including 4% Nelumbo nucifera extract was stable for 30 days under various temperature conditions. From the study, Nelumbo nucifera’s leaf, flower and seed extracts showed strong possibility for whitening and anti-wrinkle functional cosmetic agent.     

Umbelliferone aminoalkyl derivatives as inhibitors of oxidosqualene cyclases from <Emphasis Type="Italic">Saccharomyces cerevisiae,Tripanosoma cruzi</Emphasis>, and <Emphasis Type="Italic">Pneumocystis carinii</Emphasis>

A series of umbelliferone aminoalkyl derivatives, previously studied as inhibitors of squalene-hopene cyclase, were tested as inhibitors of yeast (Saccharomyces cerevisiae) oxidosqualene cyclase (OSC) and OSC from Trypanosoma cruzi and Pneumocystis carinii expressed in yeast. Enzymes from these pathogens were included in this study to provide a preliminary screening for antiparasitic activity. Tests were carried out both on cell homogenates incubated with radiolabeled oxidosqualene and on spheroplasts incubated with radiolabeled acetate. Derivatives bearing a methylallylamino group were the most effective on all of the three enzymes. The P. carinii enzyme was the most susceptible to the action of the inhibitors, with IC₅₀ values for almost all of them ranging from 0.1 to 1μM. The T. cruzi enzyme was appreciably inhibited (IC₅₀ 4–5 μM) only by derivatives bearing a methylallylaminoalkyl flexible chain. Results identify a particularly promising new family of OSC inhibitors, for the development of novel antiparasitic agents.     

Bactericidal and Antioxidant Activity of Essential Oils from <Emphasis Type="Italic">Myristica fragrans</Emphasis> Houtt and <Emphasis Type="Italic">Salvia microphylla</Emphasis> H.B.K

This study chemically characterizes and evaluates the bactericidal and antioxidant activities of essential oils from Myristica fragrans and Salvia microphylla. The essential oils were obtained by steam distillation and were subsequently subjected to analysis by GC–MS and GC. The agar diffusion test was employed to evaluate the bactericidal activity, while the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and β-carotene/linoleic acid tests were used to determine the antioxidant activity. Terpin-4-ol (14.95%), sabinene (13.07%) and γ-terpinene (11.22%) were found to be the major constituents in the essential oil of M. fragrans by gas chromatography, whereas (E)-caryophyllene (15.35%), α-eudesmol (14.06%), β-eudesmol (8.74%) and γ-eudesmol (7.64%) were encountered in the essential oil of S. microphylla. Both essential oils showed bactericidal activity, with the essential oil of S. microphylla being more efficient. The antioxidant activity of the essential oils from M. fragrans and S. microphylla were demonstrated by the β-carotene/linoleic acid test, with IC₅₀ 976 and IC₅₀ 770 μg/mL, respectively.     

Fetal Bovine Serum Concentration Affects Δ<Superscript>9</Superscript> Desaturase Activity of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Fetal bovine serum (FBS) is an important factor in the culture of Trypanosoma cruzi, since this parasite obtains and metabolizes fatty acids (FAs) from the culture medium, and changes in FBS concentration reduce the degree of unsaturation of FAs in phosphoinositides. When T. cruzi epimastigotes were cultured with 5% instead of 10% FBS, and stearic acid was used as the substrate, ∆⁹ desaturase activity decreased by 50%. Apparent K _m and V _m values for stearic acid, determined from Lineaweaver–Burk plots, were 2 μM and 219 pmol/min/mg of protein, respectively. In studies of the requirement for reduced pyridine nucleotide, ∆⁹ desaturase activity reached a maximum with 8 μM NADH and then remained constant; the apparent K _m and V _m were 4.3 μM and 46.8 pmol/min/mg of protein, respectively. The effect of FBS was observed only for ∆⁹ desaturase activity; ∆¹² desaturase activity was not affected. The results suggest that decreased FBS in culture medium is a signal that modulates ∆⁹ desaturase activity in T. cruzi epimastigotes.     

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (K_d=10⁻¹⁵ M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures—polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [¹⁴C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons ±3.3%. This protein was shown to bind avidin (M_r=16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [¹⁴C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl-CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl-CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl-CoA carboxylase-specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M_r=230,000) purified from rainbow trout liver is acetyl-CoA carboxylase with enzyme characteristics comparable to mammalian acetyl-CoA carboxylase.