Particle-free Supernatant Fraction Research Articles

Translocation to rat liver mitochondria of phosphatidate phosphohydrolase

When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.

Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: I. Limited lysosomal enzyme release and calcium uptake.

Epithelial cells isolated from rat intestine were analyzed for their responsiveness in vitro to parathyroid hormone (PTH) and to 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Criteria included determination of whether the agonists promoted extracellular liberation of lysosomal enzyme activities above control values during incubation in Ringer's solution at 22 C. PTH-augmented release of the representative hydrolase activities, cathepsin B and acid phosphatase, to the particle-free supernatant fraction of the medium was evident within 5 min of hormone treatment and was sustained in statistically significant degree for at least 30 min to greater than 20% above control levels. Basal release rarely exceeded 15% of the total cellular content of these enzymic activities. Lactate and succinate dehydrogenase activities were undetectable in the particle-free supernatant fraction under conditions of maximal hormone effect, indicating integrity of the cells and selectivity of the organellar response. Treatment of corresponding cells with 1,25-(OH)2D3 resulted in similar time of onset, magnitude, and duration of response. The most sensitive indicator of limited lysosomal labilization by either hormone was beta-N-acetyl-D-glucosaminidase activity, which underwent accentuated extracellular liberation in the presence of as little as 10(-16) M PTH or 10(-11) M 1,25-(OH)2D3, the latter eliciting a response of greater than 40% above control levels. Parathyroidectomy diminished basal release of the hydrolase activities and sensitized the intestinal cells to the action of PTH vs. preparations from intact or sham-operated animals, as judged by excess liberation of the glycosidase. Nontarget lung cells failed to respond to supramaximal levels of either hormone by the criterion of reduced latency of lysosomal hydrolases. In additional acute experiments with intestinal cells, both PTH and 1,25-(OH)2D3 promoted enhanced 45Ca2+ accumulation above control values. Collectively, these data indicate that PTH is capable of provoking direct effects on intestinal cells, similar in onset and extent to those elicited by 1,25-(OH)2D3.

Mitochondrial biosynthesis of coenzyme A

All enzymes required for the biosynthesis of CoA from pantothenic acid are present in the particle-free supernatant fraction from rat liver. We now report that also mitochondria have the capacity for biosynthesis of CoA, with 4′-phosphopantetheine as the initial precursor. Rat liver mitochondria do not contain pantothenate kinase, 4′-phosphopantothenoyl-1-synthetase or 4′-phosphopantothenoyl-1-cysteine decarboxylase. Dephospho-CoA pyrophosphorylase and dephospho-CoA kinase are present in the inner mitochondrial membrane, however, at specific activities as high as in cytosol. K m of mitochondrial dephospho-CoA kinase for dephospho-CoA is about 0.01 mmol/1, which is one order of magnitude lower than reported for the kinase from cytosol.

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue.

Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 1. Medium‐Chain Fatty‐Acid Synthesis

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.

Fatty acid synthetase complex from the insect Ceratitis capitata

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1- 14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5–6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2 × 10 5. The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants ( V and K m ) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42 ± 1-SH residues and one phosphopantetheine moiety per 5.2 × 10 5. Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0–18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

A soluble acyl-coenzyme a oxidase from the yeast Candida utilis

trans-2-Enoyl-CoA and two unidentified polar compounds were synthesized from the corresponding long-chain acyl-CoA by a particle-free supernatant fraction obtained from Candida utilis. The enzyme was unreactive toward free fatty acids but desaturated all long-chain acyl CoAs tested (14:0, 16:0, 18:1, 18:2). Molecular oxygen was the only required cofactor. Phenazine methosulfate and 2,6-dichloroindophenol did not replace the requirement for oxygen. The activity was inhibited specifically by NADPH and stimulated by linoleic acid or linolenic acid. The enzyme was not active in log phase cultures, but was detected only in stationary phase cells. Introduction of the trans-2-double bond was confirmed by gas-liquid chromatography, thin-layer chromatography, mass spectrometry, catalytic hydrogenation, oxidative cleavage, and chemical reactivity of the product toward nucleophilic addition.

Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

NON-RIBOSOMAL POLYPEPTIDE SYNTHESIS

ABSTRACT The biosynthesis of the cyclic decapeptide antibiotic, tyrocidine, was analyzed in particle-free supernatant fractions of RNase-treated homogenates of Bacillus brevis ATCC 8185. From the extracts, three complementary fractions with molecular weights of (1) 100 000, (2) 230 000, and (3) 440 000 were obtained. (1) activates and racemizes the initiating phenylalanine, (2) activates the three following amino acids, and (3) activates the last six amino acids by primary reaction with ATP to AMP-amino acid and secondary transfer to an enzyme-bound -SH. The enzymes alone fix amino acids but do not polymerize. The mixture, on sequential addition of amino acids + ATP, polymerizes first to enzyme-bound polypeptides, which after addition of all ten amino acids, cyclize. The enzymes (2) and (3) each contain one mole of 4'-phosphopantetheine which appears to act in transpeptidation. By mild autolysis and dodecyl sulphate gel electrophoresis, the polyenzymes may be split to subunits of 70 000 molecular weight, retaining only amino acid activation. In recent attempts to analyze the biosynthesis of the thyrotropic release hormone, a tripeptide made in the hypothalamus, we have met with great difficulties due to the presence of potent peptidases in brain preparations.

The metabolism of the small intestine: Dietary induction, subcellular distribution and isoenzyme pattern of some key enzymes of glucose metabolism in ovine jejunal mucosa

1. 1. Some key enzymes of glucose metabolism were determined in particle-free supernatant fractions of ovine jejunal mucosa. Hexokinase and 6-phosphogluconate dehydrogenase activities were higher in preparations from barley-fed than those from grass-fed animals. The high- K m glucokinsae activity was not detected in these preparations. 2. 2. Of the homogenate activity 40% was found to be particle bound and 47% of the particulate activity was associated with the mitochondrial fraction. 3. 3. Treatment of mucosal homogenates with Triton X-100, with and without ATP, resulted in 180 and 207% increases in soluble hexokinase activity respectively. Similar treatment of mitochondrial fractions resulted in exposure and solubilization of a latent, bound form of hexokinase. 4. 4. The ratio of type I/type II hexokinase was 7 : 3. 5. 5. Indications of particulate localization and dietary induction of glucose dehydrogenase were obtained.

A study of some enzymes of glycerolipid biosynthesis in rat liver after subtotal hepatectomy.

1. The accumulation of triglyceride in the liver remnant after subtotal hepatectomy (removal of 82% of the liver) exceeded that described for partial hepatectomy (removal of 70% of the liver). 2. Palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and diglyceride acyltransferase activities were measured in the microsomal fraction, and phosphatidate phosphohydrolase activity was measured in the particle-free supernatant fraction, prepared from the liver remnant at various times after subtotal hepatectomy. 3. The only enzyme showing a significant change in specific activity was phosphatidate phosphohydrolase. The specific activity was approximately fivefold that of the control value at 6h after operation and threefold that of the control at 10, 16 and 24h after operation. A smaller increase in the specific activity of the enzyme in sham-operated animals occurred only at 6h after operation. 4. However, at this time the total phosphohydrolase activity of the remaining liver in the sham-operated rats was approximately threefold that in hepatectomized rats. 5. Injection of actinomycin D prevented the increase in activity of phosphatidate phosphohydrolase but did not prevent the accumulation of triglyceride.

Fatty acid biosynthesis: X. Specificity for chain-termination of fatty acid biosynthesis in cell-free extracts of lactating rabbit mammary gland

1. 1. The pattern of fatty acids synthesised from [1− 14C]acetate by subcellular fractions of lactating rabbit mammary gland fortified by appropriate cofactors was found to be dependent on the concentration of the fractions as measured by protein. 2. 2. At high concentrations (14–22 mg protein/ml), the cell-free homogenate, the microsomal plus particle-free supernatant, and the particle-free supernatant fractions all synthesised a high proportion of the medium-chain fatty acids ( C 8:0) and C 10:0) which are characteristic of rabbit milk. 3. 3. Dilution of these fractions to less than 1 mg protein/ml caused a progressive increase in the proportion of long-chain acids ( C 14:0 and C 16:0) synthesised. 4. 4. With concentrated subcellular fractions, synthesis of medium-chain fatty acids was not dependent on the rate of fatty acid synthesis, the concentration of malonyl-CoA, or the amount of fatty acid synthetase present. 5. 5. Though there was a correlation between the synthesis of medium-chain fatty acids and of triglyceride by particulate fractions, esterification of synthesised fatty acids did not influence the proportion of medium-chain acids formed. 6. 6. Evidence is presented that chain termination at C 8:0 and C 10:0 acids is controlled by a factor (or factors) present in the particle-free supernatant fraction. This factor is not lost on dialysis, and can be precipitated between 40 and 100% saturation with (NH 4) 2SO 4 or by heating.

Regulation of the chain-length of fatty acids synthesized by cell-free preparations of lactating rabbit, rat and guinea-pig mammary gland

1. 1. The synthesis of medium-chain fatty acids by cell-free extracts of rabbit and rat mammary glands in proportions similar to those synthesized in vivo only occurred at high protein concentrations. The inability of subcellular fractions of guinea-pig mammary gland to synthesize medium-chain fatty acids correlates with the presence of only long-chain fatty acids in guinea-pig milk fat. 2. 2. Dilution of subcellular fractions progressively increased the proportion of long-chain fatty acids synthesized until the pattern resembled that obtained with isolated fatty acid synthetase. 3. 3. The specificity of fatty acid synthesis was not influenced by the esterification of the synthesized fatty acids into glycerides. 4. 4. Fatty acid synthetase may interact with other protein components in the particle-free supernatant fraction of rabbit and rat mammary gland, thereby altering its chain-length specificity.

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro.

The proportion of C(8:0) and C(10:0) fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.

The fatty acid synthetase complex of lactating guinea-pig mammary gland

1. 1. Fatty acid synthetase has been isolated from the particle-free supernatant fraction of homogenates from lactating guinea-pig mammary gland. 2. 2. The fatty acid synthetase complex was homogenous when analysed by gel filtration and by ultracentrifugation. The molecular weight was 4.0 × 10 5. 3. 3. The complex synthesized a range (C 4 : o−C 18 : 0) of fatty acids, though palmitate (C 16: 0) was the predominant product. The proportions of fatty acids synthesized varied with the substrate concentrations used.

The metabolism of the small intestine: Glycolysis in the mucosa of the small intestine of the sheep

1.1. Between 78–119 per cent of the glycolytic activity of homogenates of the mucosa from the jejunum of sheep was recovered in the particle-free supernatant fraction.2.2. High rates of l-lactate formation were obtained with glucose 6-phosphate as substrate; these were about two to three times those with fructose or glucose. Some l-glycerol 3-phosphate was formed from all three substrates.3.3. There was an increase in the specific activities of supernatant fractions with the age of the sheep over the period of 194 days investigated.4.4. It was not possible, in two experiments, to demonstrate any effects of different diets on the glycolytic activity of the jejunum.

Fatty acid biosynthesis V. Purification and characterisation of fatty acid synthetase from lactating-rabbit mammary gland

1. 1. Fatty acid synthetase has been isolated from the particle-free supernatant fraction of lactating-rabbit mammary gland homogenates. 2. 2. Fatty acid synthetase was homogeneous based on the criteria of sedimentation as a single symmetrical peak on analytical ultracentrifugation and elution as a single peak with constant specific activity from Sephadex G-200 and DEAE-52 cellulose. However, traces of acetyl-CoA: COa ligase (ADP) and ATP: citrate lyase were associated with the synthetase. Malonyl-CoA carboxy-lyase activity, for which a modified assay is described, co-purified with fatty acid synthetase. 3. 3. The molecular weight of the fatty acid synthetase was 9.1·10 5 and its hydrated radius 120 Å. It contained 58–59 sulphydryl groups per mole and the amino acid composition is given. 4. 4. The stability of fatty acid synthetase is described. The schlieren patterns of aged preparations showed that dissociation had occurred and this was not reversed on partial reactivation of the enzyme. 5. 5. In the absence of NADPH, fatty acid synthetase formed triacetic lactone from both acetyl-CoA and from acetyl-CoA plus malonyl-CoA. 6. 6. Using optimum substrate concentrations, fatty acid synthetase synthesised a wide range (C 4:0−C 18:0) of fatty acids. The major product was C 16:0 and a significant amount of C 4:0 was also synthesised.

The enzymic breakdown of lipids in potato tuber by phospholipid- and galactolipid-acyl hydrolase activities and by lipoxygenase

Homogenization of potato tubers at 0° results in rapid enzymic hydrolysis of the endogenous phospholipids and galactolipids to produce free fatty acids and fatty acid hydroperoxides. The enzymes responsible for these effects have been identified as acyl hydrolases and lipoxygenase. Monogalactosyl diglyceride is particularly susceptible to hydrolysis and monogalactosyl monoglyceride was detected as an intermediate product. Acyl transferase activity was also associated with the acyl hydrolase action. Properties of the various enzyme activities are described; all are present mainly in the particle-free supernatant fraction and have acidic pH optima. Evidence is presented which indicates that free lipids are hydrolysed more readily than the lipids of lipoprotein membranes. The results are discussed with particular reference to the lipid composition and enzyme activities of subcellular organelles prepared from tissue homogenates.

Biosynthesis of fatty acids in mammary tissue: I. Purification and properties of fatty acid synthetase from lactating-goat mammary tissue

Fatty acid synthetase of a high degree of purity has been prepared from the particle-free supernatant fraction of lactating-goat mammary tissue. Ultracentrifugation and polyacrylamide gel electrophoresis revealed heterogeneity of the preparation. However, the enzyme complex was sufficiently free of malonyl-CoA decarboxylase activity to enable the demonstration of the requirement for the “primer” acyl-CoA in addition to malonyl-CoA and NADPH. The enzyme appeared to utilize butyryl-CoA more efficiently than acetyl-CoA as “primer.” The nature and extent of the fatty acids synthesized using these two “primers” were characteristically different and were pronouncedly influenced by the concentration of malonyl-CoA.

Biosynthesis of fatty acids in mammary tissue: II. Synthesis of butyrate in lactating rabbit mammary supernatant fraction by the reversal of β-oxidation

An enzyme fraction has been obtained from lactating rabbit mammary particlefree supernatant fraction which synthesizes butyrate from acetyl-CoA by the reversal of β-oxidation. This fraction has been shown to contain acetoacetyl-CoA thiolase, NADH-dependent acetoacetyl-CoA reductase, and crotonyl-CoA hydratase. The reduction of crotonyl-CoA appears to be brought about by the fatty acid synthetase using NADPH or NADH. The two reducing reactions are believed to overcome the thermodynamically unfavorable reaction involved in the condensation of two molecules of acetyl-CoA.