scholarly journals Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding

1982 ◽  
Vol 203 (1) ◽  
pp. 45-50 ◽  
Author(s):  
P M Ahmad ◽  
D S Feltman ◽  
F Ahmad
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Specific Activity ◽  
Simple Procedure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Performic Acid ◽  
Mammary Tissue ◽  
Rat Mammary Gland

A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4′-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity.

scholarly journals Studies on acetyl-CoA carboxylase and fatty acid synthase from rat mammary gland and mammary tumours

1982 ◽  
Vol 208 (2) ◽  
pp. 443-452 ◽  
Author(s):  
P M Ahmad ◽  
D S Feltman ◽  
F Ahmad
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Mammary Tissue ◽  
Acetyl Coa Carboxylase ◽  
Lipogenic Enzymes ◽  
Acetyl Coa ◽  
Rat Mammary Gland ◽  
Mammary Gland Differentiation ◽  
Mammary Adenocarcinomas

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase, were determined in two transplantable mammary adenocarcinomas (13762 and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammary tissue of control animals (non-tumour-carrying) of comparable physiological states. During mammary-gland differentiation of control or tumour-carrying animals, the activities of acetyl-CoA carboxylase and fatty acid synthase in the lactating gland increased by about 40-50-fold over the values found in non-pregnant animals. On the other hand, in tumours carried by lactating dams there were only modest increases (1.5-2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with the neoplasms carried by non-pregnant animals. On the basis of the Km values for different substrates and immunodiffusion and immunotitration data, the fatty acid synthase of neoplastic tissues appeared to be indistinguishable from the control mammary-gland enzyme. However, a comparison of the immunotitration and immunodiffusion experiments indicated that the mammary-gland acetyl-CoA carboxylase might differ from the enzyme present in mammary neoplasms.

scholarly journals Regulation of lipogenic capacity in lactating rats

1982 ◽  
Vol 208 (3) ◽  
pp. 611-618 ◽  
Author(s):  
M R Grigor ◽  
A Geursen ◽  
M J Sneyd ◽  
S M Warren
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Malic Enzyme ◽  
Fatty Acid Synthase ◽  
Specific Activity ◽  
Mammary Glands ◽  
Lipogenic Enzymes ◽  
Pregnancy And Lactation ◽  
Specific Activities

1. The rate of mammary-gland lipogenesis measured in vivo from 3H2O was suppressed after decreasing the milk demand by decreasing the number of pups from ten to two or three, as well as by giving diets containing lipid [Grigor & Warren (1980) Biochem. J. 188, 61-65]. 2. The specific activities of the lipogenic enzymes fatty acid synthase, glucose 6-phosphate dehydrogenase and ‘malic’ enzyme increased between 6- and 10-fold in the mammary gland and between 2- and 3-fold in the livers during the first 10 days of lactation. The increases in specific activity coupled with the doubling of liver mass which occurred during pregnancy and lactation resulted in considerable differences in total liver activities when compared with virgin animals. 3. Although consumption of a diet containing 20% peanut oil suppressed the activities of the three lipogenic enzymes in the livers, only the ‘malic’ enzyme was affected in the mammary glands. 4. In contrast, decreased milk demand did not affect the specific activities of any of the liver enzymes, whereas it resulted in suppression of all three lipogenic enzymes of the mammary glands. There was no effect on either the cytoplasmic malate dehydrogenase or the lactate dehydrogenase of the mammary gland. 5. In all the experiments performed, the activity of the fatty acid synthase correlated with the amount of material precipitated by the rabbit antibody raised against rat fatty acid synthase.

scholarly journals Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

scholarly journals Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis

1976 ◽  
Vol 160 (3) ◽  
pp. 683-691 ◽  
Author(s):  
J Knudsen ◽  
S Clark ◽  
R Dils
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Chain Elongation ◽  
Medium Chain ◽  
Chain Acyl

1. An acyl-thioester hydrolase was isolated from the cytosol of lactating-rabbit mammary gland. The purified enzyme terminates fatty acid synthesis at medium-chain (C8:0-C12:0) acids when it is incubated with fatty acid synthetase and rate-limiting concentrations of malonyl-CoA. These acids are characteristic products of the lactating gland. 2. The mol.wt. of the enzyme is 29000±500 (mean±S.D. of three independent preparations), as estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The enzyme also hydrolyses acyl-CoA esters of chain lengths C10:0-C16:0 when these are used as model substrates. The greatest activity was towards dodecanoyl-CoA, and the three preparations had specific activities of 305, 1130 and 2010 nmol of dodecanoyl-CoA hydrolysed/min per mg of protein when 56muM substrate was used. 4. The way in which this enzyme controls the synthesis of medium-chain fatty acids by fatty acid synthetase is briefly discussed.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Regulation of triglyceride synthesis in the parturient guinea-pig mammary gland

1967 ◽  
Vol 105 (1) ◽  
pp. 225-231 ◽  
Author(s):  
N. J. Kuhn
Keyword(s):  
Fatty Acid Composition ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Milk Fat ◽  
Specific Activity ◽  
Plasma Triglyceride ◽  
Mammary Tissue ◽  
Tissue Concentrations ◽  
Fat Synthesis

1. The specific activity of the enzyme palmitoyl-CoA–l-glycerol 3-phosphate palmitoyltransferase (EC 2.3.1.15) in the mammary tissue of guinea pigs has been shown to increase 37-fold at parturition. 2. Increases also occur in tissue concentrations of glycerol 3-phosphate, CoA and free fatty acid, but not in that of acid-insoluble CoA. 3. The isolation and fatty acid composition of plasma triglyceride and of mammary-tissue free fatty acid, diglyceride and triglyceride are described. 4. The findings are discussed in relation to the regulation of milk fat synthesis.

scholarly journals Purification, Characterization, and Identification of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus

2002 ◽  
Vol 46 (5) ◽  
pp. 1310-1318 ◽  
Author(s):  
Xin He ◽  
Kevin A. Reynolds
Keyword(s):  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Genomic Sequence ◽  
Acyl Carrier Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Carrier Protein

ABSTRACT Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent Km for malonyl-ACP was 1.76 ± 0.40 μM, and the enzyme was active with acetyl-CoA (k cat, 16.18 min−1; Km , 6.18 ± 0.9 μM), butyryl-CoA (k cat, 42.90 min−1; Km , 2.32 ± 0.12 μM), and isobutyryl-CoA (k cat, 98.0 min−1; Km , 0.32 ± 0.04 μM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 μM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 ± 0.10 μM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 ± 0.08 μM).

scholarly journals Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA

1989 ◽  
Vol 17 (2) ◽  
pp. 567-586 ◽  
Author(s):  
Michael Schweizer ◽  
Kenji Takabayashi ◽  
Thomas Laux ◽  
Karl-Friedrich Beck ◽  
Rosemarie Schreglmann
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Rat Mammary Gland

Prolactin-stimulated polymerization of acetyl-CoA carboxylase in explants of mid-pregnant rat mammary gland

2001 ◽  
Vol 68 (3) ◽  
pp. 351-355 ◽  
Author(s):  
JULIUS E. OBEN ◽  
RAYMOND R. DILS
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Specific Activity ◽  
Acid Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Pregnant Rat ◽  
Rat Mammary Gland ◽  
Total Enzyme

Prolactin significantly increased the rate of fatty acid synthesis in explants of mid-pregnant rat mammary gland cultured for 96 h with insulin plus corticosterone. Under these conditions, prolactin increased the specific activity of total acetyl-CoA carboxylase in nuclear-free homogenates of explants by 2·6, and increased the proportion of the enzyme in the active polymeric form from 0·44 to 0·89. Removal of prolactin after 48 h in culture decreased the specific activity of the total enzyme by about half, and decreased the proportion as polymer to 0·52. The results show that prolactin plays a major role in mid-pregnant rat mammary gland in the polymerization which accompanies increased activity of the total enzyme and increased rate of fatty acid synthesis.

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