Fatty acid synthetase of bovine mammary: Properties and products

Fatty acid synthetase purified (20 times) from lactating bovine mammary tissue had an approximate mot wt of 485,000. The enzyme had a high content of acidic and hydrophobic amino acid residues; 62±4 sulhydryl groups and one 4′ phosphopantetheine residue/mole of enzyme. The enzyme was relatively stable when stored (3 mg/ml) in potassium phosphate buffer (250 mM), containing dithiothreitol (5 mM) at −5 C or at −30C or as a lyophilized powder at −30 C. Preincubation at 37 C in presence of dithioltreitol (5 mM) was necessary for obtaining maximum activity at the optimum pH of 6.8. Maximum specific activity of the isolated enzyme was 55 nmoles acetyl-coenzyme A min⁻¹mg⁻¹ incorporated A min⁻¹mg⁻¹ incorporated into fatty acids. Butyryl-coenzyme A or acetyl-coenzyme A (30μM), malonyl-coenzyme A (65 μM), and nicotinamide adenine dinucleotide phosphate, reduced form (300 μM) were required for optimum fatty acid synthesis. Malonyl-coenzyme A decarboxylase activity (5 nmoles min⁻¹mg⁻¹) associated with the enzyme permitted fatty acid synthesis in the presence of nicotinamide adenine dinucleotide phosphate, reduced form and malonyl-coenzyme A. The enzyme utilized acetyl-coenzyme A, butyryl-coenzyme A, and hexanoyl-coenzyme A as primers, with butyryl-coenzyme A giving the maximum rate of fatty acid synthesis. Apparent Km values of 22,6.7, 3, 22, and 20 μM were obtained for malonyl-coenzyme A, acetyl-coenzyme A, butyryl-coenzyme A, hexanoyl-coenzyme A, and nicotinamide adenine dinucleotide phosphate, reduced form, The fatty acid synthetase was inhibited by N-ethylmaleimide, iodoacetamide, and progressively inhibited by increasing concentrations of long chain acyl-coenzyme A, i.e. palmityl-coenzyme A and myristyl-coenzyme A. This inhibition was relieved by bovine serum albumin or β-lactoglobulin (3 mg/ml). Palmitic acid was the major product of bovine mammary fatty acid synthetase. However, small amounts of fatty acids, 4∶0–14∶0 inclusive, also were synthesized. The pattern of fatty acids was altered by varying malonyl-coenzyme A to acetyl-coenzyme A ratios and by increasing the enzyme levels in the assays. At high concentrations of enzyme (0.5 mg/ml), greater amounts of short and medium chain fatty acids were generated.     

Lipid peroxidation in chronic ethanol treated rats: In vitro uncoupling of peroxidation from reduced nicotine adenosine dinucleotide phosphate oxidation

Chronic ethanol treated rats were found to have enhanced ethanol metabolism and to metabolize ethanol in vivo in the presence of an inhibitor of alcohol dehydrogenase. In vitro studies of the hepatic microsomal system thought to the responsible for this activity showed it to be markedly induced. Lipid peroxidation also was enhanced in the ethanol treated animals. The lipid peroxidation was shown to be uncoupled from the microsomal nicotinamide adenine dinucleotide phosphate, reduced form, oxidase activity by a low concentration of azide.     

Electrochemical study in both classical cell and microreactors of flavin adenine dinucleotide as a redox mediator for NADH regeneration

The electrochemical reduction of flavin adenine dinucleotide (FAD) is studied in a classical electrochemical cell as well as in two types of microreactors: the first one is a one-channel reactor and the other one, a multichannel filter-press reactor. The ultimate goal is to use the reduced form of flavin (FADH₂), in the presence of formate dehydrogenase (FDH), in order to continuously regenerate the reduced form of nicotinamide adenine dinucleotide (NADH) for chiral syntheses. Various voltammetric and adsorption measurements were carried out for a better understanding of the redox behavior of the FAD as well as its adsorption on gold. Diffusivity and kinetic electrochemical parameters of FAD were determined.     

In vivo and in vitro biosynthesis of free fatty alcohols inEscherichia Coli K-12

In vivo studies have indicated that exogenous free fatty acids may serve as precursors of the free fatty alcohols ofEscherichia coli K-12. Following disruption of the cells, the enzymatic activity capable of catalyzing the reduction of long chain fatty aldehydes to fatty alcohols was localized in the 100,000 x g supernatant fraction. Nicotinamide adenine dinucleotide phosphate, reduced form, was the required cofactor. The product of the reaction was characterized rigorously as 1-hexadecanol when hexadecanal was the substrate. Three independent, but complementary, assay methods were developed to assay the aldehyde reductase activity. By employing these methods, an equivalence between nicotinamide adenine dinucleotide phosphate, reduced form, oxidation and 1-hexadecanol synthesis was established. Two protein fractions catalyzing the reduction of fatty aldehydes to fatty alcohols were detected in the 100,000 x g supernatant fraction following ammonium sulfate fractionation and diethylaminoethyl-cellulose chromatography. Enzymatic activity (70%) applied to the diethylamino-ethyl-cellulose column was eluted at a phosphate concentration of 0.115 M. The remaining 30% was eluted at a concentration of 0.23 M. Following sephadex chromatography, it was observed that the enzyme eluting at 0.115 M phosphate had an apparent mol wt of 250,000 Daltons while that eluting at 0.23 M had an apparent mol wt of 62,000 Daltons. The enzymes were similar with respect to substrate specificity, pH optima, ionic strength optima, and stability with respect to thiol inhibitors, suggesting different sized aggregates of similar subunits.     

Enzymatic determination of citrate in detergent products

A routine analytical procedure employing enzyme methodology has been developed for the determination of the citrate content of commercial detergent products. The enzyme system used is based upon the selective cleavage of citrate by citrate lyase (citrate oxaloacetate lyase; EC 4.1.3.6). One of the products, oxaloacetate, is reduced to malate by malic dehydrogenase (EC 1.1.1.37) with the simultaneous oxidation of reduced β-nicotinamide adenine dinucleotide to β-nicotinamide adenine dinucleotide, oxidized form. The course of the reaction is measured spectrophotometrically. The decrease in absorbance at 340 nm caused by the formation of β-nicotinamide adenine dinucleotide, oxidized form, is directly proportional to the concentration of citrate. The accuracy of the method which was determined by recovering citrate spikes into citrate-free detergent formulations is excellent. The precision of the method (±1.4% [two standard deviations relative to the average]) is adequate for an analysis of this type. Several commercial detergent products containing 10–20% trisodium citrate were assayed by this method.     

Induction of cell death of gastric cancer cells by a modified compound of the annonaceous acetogenin family

Annonaceous acetogenins are a family of natural products with antitumor activities. The polyether mimic AA005 has been synthesized. The cytotoxic mechanism of AA005 was investigated. It was found that AA005 can induce the cell death of gastric tumor cells. Most AA005-induced cell death is due to necrosis but partial and p53-independent apoptosis is also detected. An expanded study with gastric tumor cells and a cell-free system indicates that AA005 affects NADH:ubiquinone oxidoreductase (complex I; NADH=nicotinamide adenine dinucleotide, reduced form) in the mitochondrial electron transport system. These results show for the first time that the polyether mimic AA005 acts on the same biological target as the natural annonaceous acetogenins.     

Altered stearoyl-CoA desaturase activities in Morris hepatomas 5123C and 7800

The nicotinamide adenine dinucleotide, reduced form, dependent microsomal stearoyl-CoA desaturase activities were determined in Morris hepatomas 5123C and 7800 and compared with those of the host livers. In hepatoma 5123C, the desaturase activity was ca. one-third that of the host liver. One week on a fat-free high-carbohydrate diet did not alter the desaturase activities of the hepatomas, whereas the host liver desaturase activities increased ca. three times. Fatty acid synthetase activity of the hepatoma 5123C was ca. one-fourth and that of hepatoma 7800 was ca. the same as that of the host liver, suggesting that the decreased desaturase activity of the hepatoma 5123C is mainly due to the decreased overall lipogenesis.     

Oxidative reactions of the pentose pathway and the origin of reducing equivalents for the biosynthesis of rubber in Hevea brasiliensis latex

The presence of the oxidative reactions of the pentose phosphate pathway in hevea latex and their interaction with the biosynthesis of rubber are demonstrated using radioisotopes. During the second oxidation–reduction reaction of the pentose phosphate pathway the tritium in C₃ of glucose‐6‐phosphate is transported as tritiated nicotinamide adenine dinucleotide phosphate and serves to reduce [3‐¹⁴C] 3‐hydroxy‐3‐methylglutaryl coenzyme A to mevalonate. Reduced nicotinamide adenine dinucleotide phosphate is the inter‐connection between these two metabolic routes present in hevea latex, thus the hydrogens of glucose end up in rubber. © 2000 Society of Chemical Industry     

Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro

An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo.     

天然烟酰胺辅因子再生体系及其人工类似物研究进展

氧化还原酶可以催化具有特定区域选择性、化学选择性、立体选择性的反应,反应条件温和且催化效率高,因此在有机化学和制药领域发挥着日益重要的作用。绝大多数氧化还原酶依赖烟酰胺腺嘌呤二核苷酸NAD（H）和烟酰胺腺嘌呤二核苷酸磷酸NADP（H）为酶促反应提供氧化还原当量。NAD（H）/NADP（H）由于价格昂贵、稳定性差导致无法化学计量投入。经过几十年研究,形成了4种经典的NAD（H）/NADP（H）再生方法：酶法、化学法、电化学法和光化学法,与此同时,一系列稳定性好、活性高且廉价的人工烟酰胺辅因子mNAD（H） s尤其是1,4-二氢吡啶类烟酰胺辅因子的开发和利用为NAD（H）/NADP（H）工业化运用提供了新的思路。     

Engineering d-Lactate Dehydrogenase to Favor an Non-natural Cofactor Nicotinamide Cytosine Dinucleotide

Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d -lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a “gate” guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d -lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.     

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.     

Rewired Cellular Metabolic Profiles in Response to Metformin under Different Oxygen and Nutrient Conditions

Metformin is a metabolic disruptor, and its efficacy and effects on metabolic profiles under different oxygen and nutrient conditions remain unclear. Therefore, the present study examined the effects of metformin on cell growth, the metabolic activities and consumption of glucose, glutamine, and pyruvate, and the intracellular ratio of nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADH) under normoxic (21% O₂) and hypoxic (1% O₂) conditions. The efficacy of metformin with nutrient removal from culture media was also investigated. The results obtained show that the efficacy of metformin was closely associated with cell types and environmental factors. Acute exposure to metformin had no effect on lactate production from glucose, glutamine, or pyruvate, whereas long-term exposure to metformin increased the consumption of glucose and pyruvate and the production of lactate in the culture media of HeLa and HaCaT cells as well as the metabolic activity of glucose. The NAD⁺/NADH ratio decreased during growth with metformin regardless of its efficacy. Furthermore, the inhibitory effects of metformin were enhanced in all cell lines following the removal of glucose or pyruvate from culture media. Collectively, the present results reveal that metformin efficacy may be regulated by oxygen conditions and nutrient availability, and indicate the potential of the metabolic switch induced by metformin as combinational therapy.     

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to γ-linolenic acid in rat liver microsomes

The effects of antibodies against microsomal electron-transport components on the in vitro activity of Δ⁶-desaturation of linoleic acid to γ-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the Δ⁶-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b₅ and NADPH-cytochrome c reductase, only the antibody against cytochrome b₅ demonstrated a marked ability to inhibit the Δ⁶-desaturase activity. This evidence supports a participation of cytochrome b₅ in the Δ⁶-desaturation of linoleic acid and suggests a pathway analogous to the Δ⁹-desaturation of stearyl-CoA.     

Do rat kidney cortex microsomes possess the enzymatic machinery to desaturate and chain elongate fatty acyl-CoA derivatives?

Rat kidney cortex microsomal preparations were unable to catalyze Δ9, Δ6, and Δ5 desaturation of stearoylcoenzyme A (CoA), linoleoyl-CoA and dihomo-γ-linolenoyl-CoA, respectively. The kidney cortex microsomal fraction, however, did catalyze the malonyl-CoA dependent fatty acyl-CoA elongation. The biochemical properties of palmitoyl-CoA elongation were studied as a function of protein concentration, time, reduced nicotinamide adenine dinucleotide phosphate (NADPH), malonyl-CoA and substrate concentrations; of the substrates investigated, Δ6.9.12–18∶3 was the most active. Unlike what was observed in the hepatic system, a high-carbohydrate, fat-free diet did not induced kidney fatty acid chain elongation. All intermediate kidney cortex microsomal reactions,i.e., β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydrase andtrans-2-enoyl-CoA reductase activities, were significantly higher (greater than one order of magnitude) than the condensing enzyme activity, suggesting that the rate-limiting step in total elongation is the initial condensation reaction. Contrary to other reports, the results suggest that the kidney cannot synthesize arachidonic acid needed for eicosanoid production.     

Nicotinamide Effectively Suppresses Fusarium Head Blight in Wheat Plants

Pyridine nucleotides such as a nicotinamide adenine dinucleotide (NAD) are known as plant defense activators. We previously reported that nicotinamide mononucleotide (NMN) enhanced disease resistance against fungal pathogen Fusarium graminearum in barley and Arabidopsis. In this study, we reveal that the pretreatment of nicotinamide (NIM), which does not contain nucleotides, effectively suppresses disease development of Fusarium Head Blight (FHB) in wheat plants. Correspondingly, deoxynivalenol (DON) mycotoxin accumulation was also significantly decreased by NIM pretreatment. A metabolome analysis showed that several antioxidant and antifungal compounds such as trigonelline were significantly accumulated in the NIM-pretreated spikes after inoculation of F. graminearum. In addition, some metabolites involved in the DNA hypomethylation were accumulated in the NIM-pretreated spikes. On the other hand, fungal metabolites DON and ergosterol peroxide were significantly reduced by the NIM pretreatment. Since NIM is relative stable and inexpensive compared with NMN and NAD, it may be more useful for the control of symptoms of FHB and DON accumulation in wheat and other crops.     

Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)—thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs’ Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical’s Redox enzymatic and F-actin disassembly activity.     

Cholesterol and fatty acid synthesis in swine

In incubation studies with swine tissue slices, acetate-1-¹⁴C or glucose-U-¹⁴C as substrates were incorporated more readily into fatty acids and cholesterol in adipose tissue than other tissues tested. Cholesterol and fatty acid synthesizing activity was substantial in the small intestine. When acetate was available, liver, small intestine, and adipose tissue were important sites for cholesterol synthesis. Heart and aortic tissue had marginal levels of cholesterol synthesizing ability. Lipogenesis in adult swine liver, heart, and aortic tissue was extremely low. As in tissue slices, incorporation of acetyl-1-¹⁴C CoA into fatty acids by adipose homogenates indicated high lipogenic activity. Subcellular fractionations of heart and aortic tissue indicated that the heart microsomal fraction had the highest lipogenic activity as measured by the incorporation of acetyl-1-¹⁴C CoA into fatty acids. In adult swine adipose tissue, the incorporation of glucose-U-¹⁴C into fatty acid was higher than its incorporation into glyceride-glycerol. The synthesis of glyceride-glycerol from glucose-U-¹⁴C or acetate-1-¹⁴C in liver was higher than for fatty acid synthesis. The activity of acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, nicotinamide adenine dinucleotide phosphate-malate dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase was considerably higher in adipose tissue than in other tissues tested, paralleling its high lipogenic capacity. A preliminary report of this paper was given at the AOCS 66th Annual Spring Meeting, Dallas, Texas, April 30, 1975, Abstr. No. 109. In partial fulfillment of the requirement for the PhD degree in Nutritional Sciences at the University of Illinois at Urbana-Champaign.