The influence of environmental and incubation temperature on fatty acid synthetase from flounder (Platichthys flesus L.) liver

• 1.1. Fatty acid synthetase from liver of cold and warm adapted flounder and rabbit was purified to homogenity and compared.
• 2.2. The mol. wt of the cold and warm flounder enzyme was estimated to be about 457,000.
• 3.3. The kinetic properties were found to be similar for warm and cold adapted flounder liver enzyme and not different from the rabbit liver enzyme when measured at 5, 10, 15, 20 and 37°C.
• 4.4. Palmitic acid was the main product of both the flounder and rabbit enzyme, but significant amounts of butyric acid were also synthesized. The product composition did not change for any of the enzymes tested when the incubation temperature was changed.
• 5.5. It was concluded that fatty acid synthetase from flounder liver is similar to mammalian fatty acid synthetase with regard to molecular weight and kinetic properties.

     

The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla)

• 1.1. The extent of fatty acid synthesis from [1-¹⁴C]acetate in liver slices was reduced 6-fold when eels were fasted for 1–7 weeks and 20-fold when fasted for 39 weeks; thereafter hepatic lipogenesis seemed to remain constant for up to 95 weeks of fasting.
• 2.2. After a 1–3 week fast some hepatic enzyme activities were reduced (acetyl-CoA carboxylase decreased 2-fold and fatty acid synthetase declined 5-fold), while others remained unchanged (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, α-glycerol phosphate dehydrogenase as well as malic enzyme and ATP-citrate lyase).
• 3.3. The optimum temperature for measuring both total lipid synthesis and lipogenic enzyme activity in eel liver was found to be 30°C.

     

The effect of initiated involution on enzyme activity and substrate uptake by the mammary gland of the lactating rabbit

• 1.1. Arteriovenous difference studies across the lactating rabbit mammary gland for glucose, acetate, triacylglycerol and non-esterified fatty acids during initiated involution are reported.
• 2.2. A significant reduction in substrate utilisation is paralleled by a decrease in the activities of fatty acid synthetase, acetyl CoA synthetase, citrate synthase and glutamate dehydrogenase in biopsy samples taken from the gland.
• 3.3. Results from the analysis of lipid fractions within the gland during this period are discussed in relation to lipid resorption.

     

Effect of diet and essential amino acids gavage on young rat amino acid metabolism enzymes

• 1.1. The effects of a high-fat, high-energy diet and essential plus semi-essential amino acid gavage on pup rats have been studied (60–65 animals).
• 2.2. The activities of alanine transaminase, adenylate deaminase, glutamine synthetase and serine dehydratase have been tested in liver and muscle.
• 3.3. Plasma was used for the estimation of proteins, urea, amino acids, glucose, lactate, 3-hydroxy-butyrate and acetoacetate.
• 4.4. Liver and muscle glutamine synthetase activities are increased by diet and gavage administered. Hepatic serine dehydratase is inhibited by a cafeteria diet but activated by amino acid gavage. Adenylate deaminase is inhibited by diet and gavage in the liver, but gavage does not affect this enzyme activity in muscle. Liver alanine transaminase is increased by the diet; in the muscle, cafeteria diet and amino acid gavage showed the highest values for this enzyme.
• 5.5. In the plasma, the increase in lactate produced by the diet is inhibited by the amino acids provided. Cafeteria-fed pups showed lower urea levels and higher 3-hydroxybutyrate concentrations in the plasma.
• 6.6. Intracellular glucose is diminished by cafeteria diet. In contrast, the blood cell amino acid concentration increases with diet and gavage supplied.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Purification and immunological characterization of acid β-galactosidase from dog liver

• 1.1. Dog liver acid β-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC.
• 2.2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180–190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa.
• 3.3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated β-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat.
• 4.4. Western blot analysis of tissue extracts from dogs affected with G_MI-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.

     

Chemical characterization of the brain of a coelacanth,Latimeria chalumnae

• 1.1. The chemical composition of coelacanth brain was studied and compared with some other species of bony fishes.
• 2.2. Almost all lipid classes generally seen in vertebrate brains were detected: 22:1 and 22h:1 acids were abundant in cerebroside and 24:1 acid in ganglioside. The hydroxy fatty acid content of cerebroside was high.
• 3.3. The myelin protein composition was unusual in that a 28,000-dalton protein was a major component.
• 4.4. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase was 10 times more active than in the other bony fishes.
• 5.5. The present data suggest that molecular construction of coelacanth myelin is more advanced than that of the other bony fishes.

     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

Phospholipase A activity of culture supernatants from Streptococcus mutans strain 6715

• 1.1. Phospholipase A activity was found in the culture broth of growing cultures of Streptococcus mutans strain 6715.
• 2.2. The amount of enzyme activity was proportional to the cell density of the cultures.
• 3.3. The enzyme had a pH optimum of 7.0 and was inactivated at temperatures greater than 45°C.
• 4.4. The enzyme was Ca²⁺-dependent, since both EDTA and EGTA were inhibitory and Ca²⁺ was stimulatory.
• 5.5. Analysis of the fatty acid products resulting from the enzyme's action on 1-palmitoyl-2-oleoyl phosphatidylcholine indicated the enzyme to be a phospholipase A₁, (EC 3.1.1.32).

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

Fatty acid composition of depot fats,and rumen wall of the camel (Camelus dromedarius

• 1.1. Fatty acid composition of depot fats (hump, subcutaneous, mesentry, abdomen, perinephric and peticardiac) and rumen wall of Camelus dromedarius were studied by gas-liquid chromatography.
• 2.2. All depot fats showed similar characteristics; major fatty acids found were palmitic, stearic, oleic and myristic. Small quantities of odd-numbered, palmitoleic, linoleic and linolenic acids were also present.
• 3.3. Fatty acid of rumen wall showed some differences from the general pattern.
• 4.4. Fatty acid composition of the camel is discussed in relation to that of other ruminants and monogastrics.

     

Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

• 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
• 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
• 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
• 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
• 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent K_m of 1 mM and V_max of 0.84 μmol diene/min/mg protein.
• 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
• 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Human spleen cathepsin D: Its characterization and localization in human spleen

• 1.1. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography.
• 2.2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins.
• 3.3. Inhibition with thiol compounds was found to be a specific phenomenon of the cathepsin D from the human spleen.
• 4.4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel.
• 5.5. In an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen.
• 6.6. The role of this enzyme in human spleen cell was discussed.

     

Presence of an essential polyunsaturated fatty acid in intradigestive bacterial symbionts of a deposit-feeder echinoid (Echinodermata)

• 1.1. Fatty acids were isolated from bacteria of the family Beggiatoaceae and closely related to the genus Thiothrix. These bacteria are symbionts that live in the gut of Echinocardium cordatum.
• 2.2. Ten pronounced chromatographic peaks were observed that correspond to 14:0, 15:0, 15:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:3 and 19:0 fatty acids.
• 3.3. The fatty acid 18:3 had a retention time and mass spectrum identical to those of linolenic acid.
• 4.4. The presence of an essential fatty acid has never before been reported in a non-photosynthetic organism. This essential fatty acid in the symbiotic bacteria could be of nutritional importance for their echinoid host.
• 5.5. The presence of this essential fatty acid supports a phylogenetic affinity between Beggiatoaceae and Cyanobacteria that are the only bacteria known to synthetize linolenic polyunsaturated fatty acid (PUFA).

     

Comparison of muscle lipid composition between marine and landlocked forms of sockeye salmon (Oncorhynchus nerka)

• 1.1. Total lipid content, and lipid class and fatty acid compositions in the muscle were analyzed for marine and landlocked forms of sockeye salmon.
• 2.2. Little difference was found for the total lipid content in the muscle between both forms.
• 3.3. Triglycerides were higher in the marine form than those in the landlocked one, but phospholipids showed an opposite tendency.
• 4.4. In the fatty acid composition of total lipid, percentages of 20:1 and 22: 6n-3 were higher in the marine form, while 18:2, 18:3n-3, 18:4n-3 and 20:4n-3 were more abundant in the landlocked one. Fairly high levels of 22:1 were present only in the marine form.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.