Cholinephosphotransferase activities in microsomes and neuronal nuclei isolated from immature rabbit cerebral cortex: the use of endogenously generated diacylglycerols as substrate

1982 ◽  
Vol 60 (7) ◽  
pp. 724-733 ◽  
Author(s):  
R. Roy Baker ◽  
Huu-Yi Chang
Keyword(s):  
Phospholipase C ◽  
Microsomal Fraction ◽  
Neuronal Development ◽  
Nuclear Fraction ◽  
Neuronal Nuclei ◽  
Nuclear Membranes ◽  
Choline Phosphotransferase ◽  
Rabbit Cerebral Cortex ◽  
Low Levels ◽  
Triton X

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities were much more profoundly inhibited at low levels of Triton X-100 (0.01–0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

The acylation of 1-acyl-sn-glycero-3-phosphate by neuronal nuclei and microsomal fractions of immature rabbit cerebral cortex

1990 ◽  
Vol 68 (3) ◽  
pp. 641-647 ◽  
Author(s):  
R. Roy Baker ◽  
H.-Y. Chang
Keyword(s):  
Cerebral Cortex ◽  
Phosphatidic Acid ◽  
Lysophosphatidic Acid ◽  
Monounsaturated Fatty Acids ◽  
Acid Formation ◽  
Nuclear Fraction ◽  
Neuronal Nucleus ◽  
Neuronal Nuclei ◽  
Low Concentrations ◽  
Rabbit Cerebral Cortex

The acylation of 1-acyl-sn-glycero-3-phosphate to form phosphatidic acid was studied using a neuronal nuclear fraction N1 and microsomal fractions P3, R (rough), S (smooth), and P (neuronal microsomes from nerve cell bodies) isolated from cerebral cortices of 15-day-old rabbits. The assays contained this lysophospholipid, ATP, CoA, MgCl2, NaF, dithiothreitol, and radioactive palmitate, oleate, or arachidonate. Of the subfractions, N1 and R had the highest specific activities (expressed per micromole phospholipid in the fraction). The rates with oleate were two to four times the values seen for phosphatidic acid formation from sn-[3H]glycero-3-phosphate and oleoyl-CoA. Using oleate or palmitate, fraction R had superior specific rates to N1 at low lysophosphatidic acid concentrations. With increasing lysophospholipid concentrations the specific rates of N1 and R came closer together and maintained at least a twofold superiority over fraction P. Fraction S had the lowest specific rates of phosphatidic acid formation. Fractions N1, R, and P showed a preference for palmitate and oleate over arachidonate, particularly at low concentrations of lysophosphatidic acid. For N1 and R, the preference was also more marked at higher concentrations of fatty acid. Thus a selectivity for saturated and monounsaturated fatty acids was shown in the formation of phosphatidic acid, as was a concentration of acylating activity in the neuronal nucleus and the rough endoplasmic reticulum.Key words: 1-acyl-sn-glycero-3-phosphate, acylation, neuronal nuclei, microsomes, cerebral cortex.

scholarly journals Studies on the sterols and sterol esters of the intracellular organelles of maize shoots

1968 ◽  
Vol 110 (1) ◽  
pp. 119-125 ◽  
Author(s):  
R. J. Kemp ◽  
E. I. Mercer
Keyword(s):  
Linolenic Acid ◽  
Microsomal Fraction ◽  
The Other ◽  
Nuclear Fraction ◽  
Unesterified Cholesterol ◽  
Sterol Esters ◽  
Mitochondrial Fractions ◽  
Intracellular Organelles

1. The composition of the esterified and unesterified sterols of the nuclear, chloroplastidic, mitochondrial and microsomal fractions of 21-day-old maize shoots was examined. 2. The microsomal and mitochondrial fractions contain the bulk of the sterols of the tissue. 3. Only 1% of the sterol isolated from all the organelles is esterified. 4. The nuclear fraction has the greatest proportion of esterified sterol and the microsomal fraction the least. 5. 4-Demethyl sterols constitute the bulk of both esterified and unesterified sterols in all organelle fractions. 6. Cholesterol is the major esterified 4-demethyl sterol of the nuclear and chloroplastidic fractions, but only the nuclear fraction has an appreciable proportion of unesterified cholesterol. 7. Sterol esters of linolenic acid are more abundant in the mitochondrial and microsomal fractions than in the other two fractions.

Conjugates of ubiquitin cross-reactive protein distribute in a cytoskeletal pattern

1994 ◽  
Vol 14 (12) ◽  
pp. 8408-8419 ◽  
Author(s):  
K R Loeb ◽  
A L Haas
Keyword(s):  
Cell Lines ◽  
Intermediate Filaments ◽  
Enzymatic Reaction ◽  
Mesothelial Cell ◽  
A549 Cells ◽  
Reactive Protein ◽  
Cell Extracts ◽  
Intracellular Proteins ◽  
Low Levels ◽  
Triton X

Ubiquitin cross-reactive protein (UCRP), a 15-kDa interferon-induced protein, is a sequence homolog of ubiquitin that is covalently ligated to intracellular proteins in a parallel enzymatic reaction and is found at low levels within cultured cell lines and human tissues not exposed to interferon. Ubiquitin and UCRP ligation reactions apparently target distinct subsets of intracellular proteins, as judged from differences in the distributions of the respective adducts revealed on immunoblots. In this study, successive passages of the human lung carcinoma line A549 in the presence of neutralizing antibodies against alpha and beta interferons had no effect on the levels of either free or conjugated UCRP, indicating that these UCRP pools are constitutively present within uninduced cells and are thus not a consequence of autoinduction by low levels of secreted alpha/beta interferon. In an effort to identify potential targets for UCRP conjugation, the immunocytochemical distribution of UCRP was examined by using affinity-purified polyclonal antibodies against recombinant polypeptide. UCRP distributes in a punctate cytoskeletal pattern that is resistant to extraction by nonionic detergents (e.g., Triton X-100) in both uninduced and interferon-treated A549 cells. The cytoskeletal pattern colocalizes with the intermediate filament network of epithelial and mesothelial cell lines. Immunoblots of parallel Triton X-100-insoluble cell extracts suggest that the cytoskeletal association largely results from the noncovalent association of UCRP conjugates with the intermediate filaments rather than direct ligation of the polypeptide to structural components of the filaments. A significant increase in the sequestration of UCRP adducts on intermediate filaments accompanies interferon induction. These results suggest that UCRP may serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments.

Purification par séparation de phase et caractérisation du plasmalemme de l'épicotyle de pois

1986 ◽  
Vol 64 (5) ◽  
pp. 448-455 ◽  
Author(s):  
Jacques Rembur ◽  
Pierre Landré ◽  
Arlette Nougarède
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Alkaline Ph ◽  
Phase Partition ◽  
Pea Epicotyl ◽  
Glucan Synthetase ◽  
Triton X ◽  
Two Sides ◽  
Slight Contamination

The validity of phase partition to obtain a substantial proportion of vesicles of plasmalemma origin from the microsomal fraction of pea epicotyl has been demonstrated. In the fractions enriched with plasma membranes, N-naphthyl phtalamic acid binding and β-glucan synthetase II activity, showed a yield of about 60% and an enrichment of 2.3 and 2.2, respectively, in comparison with the microsomal fraction. When such plasmalemmic vesicles are permabilized by Triton X-100, an intense Mg2+-ATPase activity is obtained in presence of K+ at acid as well as alkaline pH. Inhibition of Mg2+-ATPase by vanadate in presence of K+ and its variations in relation to pH were shown. Dicyclohexylcarbodiimide and diethylstilbestrol inhibit 40–55% of this enzymatic activity, both at acid and neutral pH. The data show a slight contamination of the plasmalemmic fraction by endomembranes and suggest an asymmetry of the two sides of the plasmalemma.

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

1990 ◽  
Vol 68 (9) ◽  
pp. 1112-1118 ◽  
Author(s):  
Lee Kihn ◽  
Dorothy Rutkowski ◽  
Robert A. Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Human Liver ◽  
Red Cell ◽  
Osteosarcoma Cells ◽  
Alkaline Phosphatases ◽  
Membrane Anchor ◽  
Gradient Gel Electrophoresis ◽  
Triton X ◽  
Phosphatidylinositol Phospholipase C

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 × slower in liposomes and 100 × slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.Key words: alkaline phosphatase, liposome, phosphatidylinositol, membrane anchor.

The polypeptides of rat liver nuclear envelope. II. Comparison of rat liver nuclear membrane polypeptides with those of the rough endoplasmic reticulum

1980 ◽  
Vol 43 (1) ◽  
pp. 269-277
Author(s):  
J.C. Richardson ◽  
A.H. Maddy
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Relative Proportion ◽  
Membrane System ◽  
Membrane Systems ◽  
Nuclear Membranes ◽  
Cytoplasmic Surface ◽  
Triton X

Nuclear envelopes are separated into pore-lamina and membrane sub-fractions by extraction in 2.0% Triton X-100 followed by pelleting of the pore-laminae. The polypeptides of these subfractions are then compared with those from isolated rough endoplasmic reticulum. The dispositions of individual polypeptides in the cytoplasmic surface of nuclear envelopes and rought endoplasmic reticulum were studied by lactoperoxidase-catalysed iodination. These studies show that although the nuclear membranes exhibit several homologies with the Triton-soluble polypeptides of the rough endoplasmic reticulum the relative proportion of individual polypeptides within the two systems are very largely different. The cytoplasmic surfaces of the 2 membrane systems show only 2 obvious homologies at 105 000 and 15 000 mol. wt and the overall impression is that, at least in rat liver, the outer nuclear membrane is very substantially differentiated from rough endoplasmic reticulum. It is concluded that the nuclear membranes may not be regarded as a mere continuum of the endoplasmic reticulum, but should be seen as a highly specialized membrane system in their own right.

scholarly journals Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation

1981 ◽  
Vol 198 (1) ◽  
pp. 85-90 ◽  
Author(s):  
C T Teng ◽  
C S Teng
Keyword(s):  
Microsomal Fraction ◽  
High Mobility ◽  
Quantitative Change ◽  
High Mobility Group ◽  
Nuclear Fraction ◽  
Hmg Proteins ◽  
High Mobility Group Protein ◽  
Group Protein ◽  
Oviduct Cell

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.

scholarly journals Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

1996 ◽  
Vol 16 (10) ◽  
pp. 5857-5864 ◽  
Author(s):  
J Han ◽  
P Sabbatini ◽  
E White
Keyword(s):  
Apoptotic Cell ◽  
Yeast Two Hybrid ◽  
Nuclear Membranes ◽  
Induction Of Apoptosis ◽  
Low Levels ◽  
Apoptosis Inhibitor ◽  
Apoptosis Proteins ◽  
Two Hybrid

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.

The Triton X-100 and High Salt Resistant Residue of Saccharomyces cerevisiae Nuclear Membranes

1982 ◽  
Vol 37 (10) ◽  
pp. 916-920 ◽  
Author(s):  
Karlheinz Mann ◽  
Dieter Mecke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
Initial Treatment ◽  
High Salt ◽  
Isolated Nuclei ◽  
Nuclear Membranes ◽  
Higher Eukaryotes ◽  
Triton X

Abstract Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease after an initial treatment of nuclei with very diluted buffers. When the nuclear membranes were treated with 5% Triton X-100 and 1 ᴍ NaCl an insoluble fibrous net was obtained which consisted mainly of protein with Mr values of 85000, 48000, 45000, 39000 and 31000. Lamins, a set of proteins with Mr = 65000-75000, which were shown to be the major proteins of the insoluble nuclear membrane residue of higher eukaryotes, were not found.

scholarly journals Differential expression of the PEA3 subfamily of ETS transcription factors in the mouse ovary and peri-implantation uterus

Reproduction ◽  
2005 ◽  
Vol 129 (5) ◽  
pp. 651-657 ◽  
Author(s):  
Tae Bon Koo ◽  
Haengseok Song ◽  
Irene Moon ◽  
Kyuyong Han ◽  
Chen Chen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Neuronal Development ◽  
Early Event ◽  
Corpora Lutea ◽  
Placental Development ◽  
Mouse Ovary ◽  
Mouse Uterus ◽  
Specific Expression ◽  
Low Levels ◽  
Spatio Temporal

The objective of the present investigation was to examine the spatio-temporal expression of three members of the ETS family of transcription factors, ERM, ER81, and PEA3, in the peri-implantation mouse uterus and in the ovary. These three factors belong to the PEA3 subfamily and are known to mediate diverse functions ranging from neuronal development to tumor progression. As transcription factors, they regulate the expression of a number of genes with various biological functions. Since several genes with known roles in the reproductive processes have been shown to be under the regulation of one of these factors, we sought to investigate the expression of ERM, ER81, and PEA3 in the mouse ovary and uterus. Quantitative RT-PCR analyses showed that ERM, ER81, and PEA3 were all expressed in the peri-implantation mouse uterus, with higher levels of expression on days 4 and 5 of pregnancy. To determine the cell type-specific expression of these factors, we employedin situhybridization, the results of which revealed that ERM was expressed in both the epithelium and the stroma on days 4 and 5 of pregnancy. Uterine glands showed a high expression of ERM on those days. ERM was also highly expressed in the corpora lutea of the mouse ovary. Both ER81 and PEA3 were expressed at low levels in the stroma on days 4 and 5. On day 8, while ERM and PEA3 were mainly expressed in the embryo and were at low levels in the maternal decidua in a diffused pattern, ER81 was highly expressed in the vascular bed of the mesometrial deciduum. Both ER81 and PEA3 were undetectable in the mouse ovary. Collectively, these data show that ERM is implicated in the early event of implantation as well as in ovarian functions, while ER81 is involved in the establishment of the maternal vasculature for subsequent placental development. PEA3 is apparently an embryonic factor for early embryogenesis.

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