Postmicrosomal Supernatant Research Articles

Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes.

Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.

Protein synthesis in rat brain following neonatal exposure to lead

1. (1) Suckling rats were exposed to lead through the milk of their dams who received a diet of 4% lead carbonate and weanling rats were exposed to 2 injections of 5.0 mg Pb 2+ 100 g body weight. The brains were used to prepare the following homogenate fractions: postmitochondrial supernatant, postmicrosomal supernatant, ribosomes, initiation factors. 2. (2) The postmitochondrial supernatant fractions were tested in vitro for protein synthesizing activity using the incorporation of labelled phenylalanine, and phenylalanyl-tRNA into peptide. The preparations from the lead-exposed rats had a significant reduction in activity. 3. (3) Peptide formation with the brain ribosomes was not changed in the lead-exposed rats. 4. (4) The aminoacyl-tRNA synthetase reaction was significantly reduced and accounted for most of the reduced peptide formation with brain homogenates from lead-exposed rats. 5. (5) The binding of methionyl-tRNA fMet to ribosomes was increased using initiation factor preparations from the brain of lead-exposed rats.

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations.

The pool size and synthesis of phosphatidic acid derived from [2-3H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-3H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation: labeling decreased rapidly thereafter. In drug-treated retinas, 0.5 mM propranolol increased the endogenous content of phosphatidic acid and stimulated [2-3H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated, and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.

Subcellular distribution of alpha-chaconine in mouse hepatocytes.

alpha-Chaconine is a toxic steroidal alkaloid present as a glycoside in potatoes. alpha-Chaconine was previously reported to bind with cell homogenates of various tissues. The extent of binding of 3H-alpha-chaconine in various macromolecular and subcellular fractions of liver after oral administration in female Swiss-Webster mice was investigated. No binding of this alkaloid was noted with isolated DNA or RNA fractions. The proportion of bound activity increased as the time between dosing and sampling increased, although the total alkaloid present in the organelles decreased. The extent of binding did not change when the doses ranged between 1 and 10 mg/kg. Induction of liver microsomal enzymes by treatment with dibenzo[a]pyrene increased the percent binding in all fractions except the proteins in the post-microsomal supernatant. Similar treatment with Aroclor 1254 and phenobarbital failed to produce increased binding in any of the fractions.

Subcellular localization in normal and vitamin A-deficient rat liver of vitamin A serum transport proteins, albumin, ceruloplasmin and class I major histocompatibility antigens

The subcellular localization in rat liver cells of retinol-binding protein (RBP), prealbumin, ceruloplasmin, albumin, and class I transplantation antigen chains was investigated by radioimmunoassay determinations. The concentration of RBP was high in the rough and smooth endoplasmic reticulum (SER). The relative concentrations of prealbumin, ceruloplasmin and albumin were similar in the endoplasmic reticulum fractions and in the Golgi fraction. Neither of the proteins were found in significant amounts in the post-microsomal supernatant nor in the plasma membrane. The concentrations of the class I transplantation antigen chains were higher in the Golgi fraction than in the endoplasmic reticulum fractions. In the rough endoplasmic reticulum (RER) fraction ceruloplasmin and the class I antigens partially interact with high-molecular weight (MW) components, presumably membrane-bound glycosyltransferases. RBP, prealbumin and albumin seemed to be present in free form within the microsomal lumen. In vitamin A deficiency the RBP and to a lesser extent the prealbumin concentrations in the endoplasmic reticulum fractions were significantly increased, as compared to fractions from normal livers. This suggests that the presence of vitamin A is a prerequisite for the transport of RBP from the endoplasmic reticulum to the Golgi complex. The intracellular concentrations of albumin and ceruloplasmin were not significantly altered by vitamin A deficiency. In contrast, the amounts of the class I antigen heavy chains were found to be increased.

Secretory protein synthesis in parotid glands of young and old rats

To determine whether the synthesis of secretory proteins changes with age, the incorporation of [ 14C]-leucine into exportable proteins of the parotid gland was compared in 2- and 24-month-old rats. The proteins were separated by SDS-gel electrophoresis of the post-microsomal supernatant and identified by comparing the banding patterns in gels prepared from unstimulated glands with those from the glands stimulated to secrete. The amount of radioactivity incorporated into the bands corresponding to exportable proteins was significantly less in the older group, indicating that the synthesis of secretory proteins declines with age.

Identification of a high molecular weight alkaline protease in rat heart

We have identified in soluble extracts of rat heart, a 500 000 dalton sulfhydryl-dependent protease which degrades globin and casein to acid-soluble peptides at an alkaline pH optimum. This enzyme was purified more than 1700-fold with respect to the postmicrosomal supernatant. On the basis of various catalytic and biochemical properties the enzyme appears similar to a recently described cytoplasmic protease in rat liver. Protease activity in vitro was stimulated up to 3-fold by physiologic concentrations of ATP and to a lesser extent by some other phosphate-containing compounds. Unlike some alkaline proteases reported in heart tissue, this high molecular weight protease was identified in extracts from isolated cardiac myocytes and in extracts from hearts of rats treated with the mast cell degranulating agent Compound 48 80 . Thus, the identification of the protease in heart does not appear to be accounted for by mast cell contamination.

Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro.

In the preceding papers, we demonstrated that the endogenous phosphorylation of a 29,000-dalton protein is stimulated in response to secretagogue application to intact cells from the rat exocrine pancreas and parotid and dephosphorylated upon termination of secretagogue action. One- and two-dimensional gel analysis of 32Pi-labeled pancreatic and parotid lobules as well as their respective subcellular fractions revealed that the same protein was covalently modified in both tissues and was localized to the ribosomal fraction. To identify the intracellular second messengers which may mediate or modulate the phosphorylation of the 29,000-dalton protein in intact cells, the effects of Ca2+, cAMP, and cGMP on the endogenous phosphorylation of this protein were assessed in subcellular fractions from the rat pancreas and parotid. Our results demonstrate that the phosphorylation of the 29,000-dalton polypeptide may be regulated by both Ca2+ and cAMP in the pancreas and in the parotid. No cGMP-dependent protein phosphorylation was found in either tissue. As in the in situ phosphorylation studies, the Ca2+- and cAMP-dependent phosphorylation of this same protein was localized to the ribosomal fraction. The cAMP-dependent protein kinase activity was found primarily in the postmicrosomal supernatant in contrast to the Ca2+-dependent protein kinase that appeared to be tightly associated with the substrate in addition to being present in the postmicrosomal supernatant. The data suggest that, in cells from the exocrine pancreas and parotid, secretagogues may regulate the phosphorylation of the 29,000-dalton protein through Ca2+ and/or cAMP.

Oxidation of corticosteroids to steroidal 20-hydroxy-21-oic acids by mouse liver

We have studied the enzyme catalyzed oxidation of 11-deoxycorticosterone to 20-hydroxy-3-oxo-4-pregnen-21-oic acid (pregnolic acid) in mouse liver. Enzyme activity, though variable, was higher than that of other species. Pregnolic acid was identified as the free acid. as the methyl ester by thin layer chromatography, and as the p-bromophenacyl ester by high performance liquid chromatography. With [4- 14C, 21- 3H]-DOC as substrate, exchange of tritium with water (interpreted as due to the reversible isomerization of the ketol to aldol form of the side chain) and the overall conversion of the ketol side chain to hydroxy acid was catalyzed by the post-microsomal supernatant fraction. Although we could not physically separate tritium exchange and acid production, pregnolic acid formation could be decreased or eliminated while tritium exchange was retained, consistent with our previous conclusion that isomerization to aldol was a precondition for acid formation. In preparations that made no acid, [4- 14C]-DOC was recovered, depleted of tritium. The rate of exchange of [21S, 21- 3H]-DOC with water was faster than [21R, 21- 3H]-DOC. The stereochemistry of pregnolic acid at C-20 was 85–90% R (i.e. 20α-hydroxy-21-oic acid). The K m for isomerase with [21RS-21 3H]-DOC was 4.3 × 10 −5M; K m for pregnolic acid formation was 8.0 × 10 −5 M. Corticosterone was oxidized to acid metabolites at 20% the rate of DOC.

Oxidation of cortisol to hydroxy acid metabolites by liver cytosol

Cytosols (post-microsomal supernatants) prepared from rat, hamster and mouse livers oxidized Cortisol to 11β,17,20-trihydroxy-3-oxo-pregn-4-en-21-oic acids. Mouse liver enzymes yielded over 90% 20α-hydroxy epimer from cortisol. 21-dehydrocortisol (11α,17-dihydroxy-3,20-dioxo-pregn-4-en-21-aldehyde), and 20α-isocortisol (11α,17,20α-trihydroxy-3-oxo-pregn-4-en-21-aldehyde). The 20β-epimer of isocortisol yielded both 20α- and 20β-hydroxy acid. Rat and hamster liver cytosols converted cortisol, 21-dehydrocortisol and 20α-isocortisol to both 20α- and 20β-hydroxy acids, with the former predominant. The hamster enzyme oxidized 20β-isocortisol mainly to the 20β-hydroxy acid. The results support our conclusion that both 17α-hydroxy and 17-deoxy corticosteroids are oxidized to hydroxy acids by similar pathways and that isosteroids are obligatory intermediates.

Membrane lipids composition and metabolism during early embryonic development phospholipid subcellular distribution and 32P labelling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented 7% of the phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gatrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.

Changes in the synthesis of exportable and nonexportable proteins in parotid glands during aging

The age-related differences in the synthesis of exportable and nonexportable proteins of the parotid salivary gland were compared in 2- and 24-month-old rats. Parotid slices from these rats were incubated in the presence of [ 14C]leucine and the amount of radioactivity incorporated into the water-soluble proteins of the postmicrosomal supernatant was compared. The exportable and nonexportable proteins were identified by electrophoretic separation of these proteins by comparing the banding patterns of the gel preparations from unstimulated glands to those from the glands stimulated to secrete. The radioactivity determination in various protein bands from these rats indicated that the synthesis of exportable secretory proteins declined with age, while that of nonexportable proteins did not appear to change.

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.

Increase in protein synthetic activity in chicken muscular dystrophy.

An increased level of protein synthesis was observed in an in vitro translation when the polyribosomes and postmicrosomal supernatant were prepared from dystrophic rather than normal muscle. This increment in protein synthetic activity was observed even in young dystrophic muscle in which no clinical and morphological abnormalities were detected. No specific increase in the rate of synthesis of any protein could be demonstrated in dystrophic muscle relative to normal muscle. Since a possible reason for the increment in protein synthetic activity was an increase in the activity of factor(s) in the postmicrosomal fraction, several of these were prepared from normal and dystrophic breast muscles and their activities were compared. Crude elongation factor (EF) from dystrophic muscle activated poly(U) directed polyphenylalanine synthesis more than the normal EF. This increase was shown to be due to higher levels of EF1 and EF2 in dystrophic than in normal muscle and not to a decrease in protease of RNase activity in the supernatant of dystrophic muscle relative to normal muscle. A comparison of the abilities of the postmicrosomal supernatants of normal and dystrophic muscle was made to support protein synthesis in vitro as a function of development age. The activity of aminoacyl-tRNA synthetase was also higher in the dystrophic supernatant than in the normal supernatant.

Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

Three forms of DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] were partially purified from the combined nuclear extract and postmicrosomal supernatant solution of synchronized HeLa cells. These enzymes, designated DNA polymerases alpha 1, alpha 2, and alpha 3, on the basis of their order of elution from DEAE-Bio-Gel, differ in their abilities to utilize single-strand DNA templates. DNA polymerase alpha 2 has equal catalytic activities with activated and single-strand DNAs as template-primers. DNA polymerase alpha 1 has only partial catalytic activity with single-strand DNA templates, and DNA polymerase alpha 3 is essentially inactive with this template. Successive steps of hydrophobic affinity chromatography and phosphocellulose chromatography of DNA polymerase alpha 2 resolved the polymerase alpha activity and two protein factors (C1 and C2) that are required for its catalytic activity with a DNA template-primer that contains extended single-strand regions. In the absence of the factors, DNA polymerase alpha activity is measurable with activated but not single-strand DNA templates. In the presence of the C1 and C2 factors DNA polymerase alpha activity with single-strand DNA templates is restored to about 75% of the catalytic activity of DNA polymerase alpha 2 with this template.

The intracellular distribution and antiviral activity of amantadine

The uptake and intracellular distribution of amantadine were examined to better define the site and mechanism of action of this drug. Amantadine is rapidly concentrated in lysosomes and in the cytosol (postmicrosomal supernatant) of Madin Darby canine kidney (MDCK) cells. A considerable portion of the cell-associated amantadine resists elution and remains in the lysosomes and the cytosol when the cells are subsequently washed and incubated in amantadine-free medium. In contrast, the antiviral activity of amantadine is rapidly lost when the drug is removed from the extracellular medium. These results indicate that most of the cell-bound amantadine does not contribute to the antiviral action of the drug. That portion of the cell-associated amantadine which does account for its antiviral activity is rapidly exchanged with the extracellular medium, and in fact, may actually be the drug in the extracellular medium.

Digestive end products mobilize secretory proteins from subcellular stores in the pancreas.

Two digestive end products, D-glucose and L-lysine, produced substantial concentration-dependent release of amylase and trypsinogen, respectively, from subcellular storage pools into a postmicrosomal supernatant fraction of rat pancreatic tissue homogenate. This process was selective in that D-glucose did not lead to trypsinogen release, while L-lysine did not effect amylase. An analogue of D-glucose, 2-deoxy-D-glucose, was much less potent than D-glucose on an equimolar basis. Half-maximal release for both end-product enzyme pairs occurred at concentrations within the range of normal plasma values for these end products in the rat. Although amylase release reached an apparent plateau when the concentration of glucose was increased beyond the maximally effective level, lysine concentrations higher than that maximally effective resulted in a fall in trypsinogen release that ultimately returned (at 3.0 mM L-lysine) to the level seen in its absence. When isolated zymogen granules were exposed to the same concentrations of D-glucose or L-lysine, a similar pattern of release was seen, indicating that the zymogen granules are a source of the enzymes released from the particulate phase of the homogenates. These findings can be explained most simply by the selective movement of digestive enzymes across zymogen granule membranes in response to the presence of appropriate end products. They are also consistent with the concept that digestive end products can act rapidly and directly on the pancreatic acinar cell to regulate the mixture of enzymes secreted in response to the specific hydrolytic needs of a meal.

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis.

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.

Comparison of 45Ca 2+ uptake activity by microsomes from control and stimulated mouse pancreatic acini

The ability of microsomal preparations to transport 45Ca 2+ was studied in preparations of control and secretagogue-stimulated pancreatic acini. ATP-dependent 45Ca 2+ uptake activity was present in the pancreatic post-mitochondrial supernatant and microsomes but little activity was present in the postmicrosomal supernatant. Treatment of acini with the secretagogues cholecystokinin (CCK) and carbamylcholine (CCh) prior to cell fractionation increased the subsequently measured microsomal 45Ca 2+ uptake. The effect of CCK was maximal after 10 min stimulation and at a not. The effect of CCK was maximal after 10 min stimulation and at a concentration of 1 nM; these conditions are comparable to the effects of CCK on 45Ca 2+ fluxes in intact acini. The increased microsomal 45Ca 2+ uptake induced by CCK was due to an increase in the maximal rate of 45Ca 2+ uptake as there was no effect on the K m for Ca 2+ (1 μM). It is concluded that secretagogues increase the ATP-dependent uptake of 45Ca 2+ by an isolated pancreatic microsomal component under the same conditions that also stimulate both digestive enzyme secretion and bi-directional Ca 2+ movements.

The metabolism of drugs and carcinogens in isolated subcellular fractions of Drosophila melanogaster. II. Enzyme induction and metabolism of benzo[ a]pyrene

The effect of various pretreatments on the activities of several drug metabolizing enzymes was investigated in microsomes and postmicrosomal supernatant fractions isolated from whole body homogenates of Drosophila melanogaster larvae of different strains. Pretreatments of larvae with either phenobarbital (PB), β-naphthoflavone (BNF) or a mixture of polychlorinated biphenyls (Aroclor 1254, PCB) for 24 h increased microsomal benzo[ a]pyrene (BP) monooxygenase activity 2- to 6-fold in all strains as compared to untreated larvae. A simultaneous increase in the contents of cytochrome P-450 occurred after pretreatment with PB and PCB. Comparison of the turnover rates of BP per molecule of cytochrome P-450 indicated that BP was a poor substrate for control cytochrome P-450 whereas BNF induced a most active hemoprotein for this metabolism. Marked differences in the qualitative pattern of BP metabolites were obtained between microsomes isolated from BNF-treated larvae or rat liver microsomes. 3-Hydroxy-BP (3-OH-BP) was the dominating metabolite with both preparations, while the BP dihydrodiols were formed in minor quantities in Drosophila as compared to rat liver. Metyrapone and SKF 525-A inhibited BP metabolism in microsomes isolated from untreated and BNF treated larvae of all strains. In contrast, α-naphthoflavone (ANF) stimulated the BP monooxygenase activity of microsomes isolated from untreated larvae approx. 3-fold but only slightly influenced the activity of microsomes from BNF treated larvae indicating that the latter species of cytochrome P-450 was less sensitive to ANF. In all strains, PCB and PB treatments approximately doubled microsomal epoxide hydrolase activity and increased cytosolic glutathione- S-transferase activity 25–60%, significant only in strain Berlin K after PB treatment. The activities of epoxide hydrolase and glutathione- S-transferase in control larvae were comparable in the different strains, whereas the content of cytochrome P-450 and BP monooxygenase activity was higher in the Hikone R strain. Variability in the induction response to the various pretreatment was observed among the three strains.