scholarly journals The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1979 ◽  
Vol 180 (3) ◽  
pp. 515-522 ◽  
Author(s):  
James Wilson ◽  
Arnaldo Corti ◽  
Margaret Hawkins ◽  
H. Guy Williams-Ashman ◽  
Anthony E. Pegg
Keyword(s):  
Rat Liver ◽  
Homoserine Lactone ◽  
Adenosylmethionine Decarboxylase ◽  
Constant Rate ◽  
14Co2 Production ◽  
Membrane Bound ◽  
Lag Period ◽  
High Concentrations ◽  
Triton X

1. The production of 14CO2 from S-adenosyl[carboxyl-14C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of 14CO2 could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56–69]. 2. The formation of 14CO2 by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5′-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce 14CO2 when incubated with [U-14C]-homoserine, and 14CO2 production was greater when S-adenosyl[carboxyl-14C]methionine that had been degraded by heating at pH6 at 100°C for 30min (a procedure known to produce mainly 5′-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-14C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that 14CO2 is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-14C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5′-methylthioadenosine and homoserine lactone.

Intracellular distribution of lead in Tetrahymena during continuous exposure to the metal

1979 ◽  
Vol 39 (1) ◽  
pp. 383-396
Author(s):  
J.R. Nilsson
Keyword(s):  
Cell Growth ◽  
Growth Medium ◽  
Food Vacuole ◽  
Continuous Exposure ◽  
Organic Growth ◽  
Food Vacuoles ◽  
Entire Cell ◽  
Membrane Bound ◽  
Lag Period ◽  
High Concentrations

Lead acetate (0.1–0.2%) forms a precipitate with the organic growth medium. The Tetrahymena cells ingest this lead-containing precipitate and cell growth is resumed after a variable lag period. Ingested lead is observed as electron-dense material in food vacuoles. Soon after exposure, cytoplasmic lead (preserved with certain fixation only) is revealed as electron-dense particles in cilia and in a halo around digestive vacuoles. Later the lead particles pervade the entire cell but after the lag period they are confined to membrane-bound spaces. In dilute growth medium, high concentrations of lead inhibit food-vacuole formation and cell growth. Under these conditions lead is deposited in alveoli of the pellicle and is also found in autophagic vacuoles and other membrane-limited structures. The study has revealed that lead enters Tetrahymena through the membrane of digestive vacuoles and through the cell surface. The change in distribution of lead during the lag period indicates that a mechanism is activated for removal of lead into membrane-bound spaces. The final storage of lead seems to be in lysosomes.

scholarly journals Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum

1976 ◽  
Vol 156 (2) ◽  
pp. 347-355 ◽  
Author(s):  
I H Fraser ◽  
S Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
High Speed ◽  
Broad Band ◽  
Chromatographic Technique ◽  
Appreciable Effect ◽  
Serum Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a ‘high-speed’ supernatant. The ‘high-speed’ supernatant was designated the ‘saline wash’ and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 × 10(-6) M. α-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

1981 ◽  
Vol 59 (7) ◽  
pp. 500-510 ◽  
Author(s):  
Paul G. Casola ◽  
Fred Possmayer
Keyword(s):  
Phosphatidic Acid ◽  
Initial Period ◽  
The Other ◽  
Rat Lung ◽  
Dependent Activity ◽  
Order Of Magnitude ◽  
Membrane Bound ◽  
High Concentrations ◽  
Triton X ◽  
Hydrolysis Of

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5–10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10–20 min), the rates were comparable.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram

1993 ◽  
Vol 292 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Y Shakur ◽  
J G Pryde ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Thermal Inactivation ◽  
Cytosol Fraction ◽  
Terminal Domain ◽  
Low Concentrations ◽  
Membrane Bound ◽  
High Concentrations ◽  
Membrane Components ◽  
Triton X

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a ‘hypotonic shock’ treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

scholarly journals Factors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration, organelle integrity and ATP

1980 ◽  
Vol 190 (3) ◽  
pp. 485-494 ◽  
Author(s):  
J Thomas ◽  
L J Debeer ◽  
P J De Schepper ◽  
G P Mannaerts
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Structural Damage ◽  
Oxidation Products ◽  
Acid Oxidation ◽  
H2o2 Production ◽  
Beta Oxidation ◽  
High Concentrations ◽  
Triton X ◽  
Peroxisomal Fatty Acid

1. The first dehydrogenation step of peroxisomal beta-oxidation involves the reduction of O2 to H2O2. Production rates of H2O2 and acetyl units by purified rat liver peroxisomes oxidizing palmitoyl-CoA were equal, indicating that H2O2 production is a reliable index for the release of acetyl units during peroxisomal fatty-acid oxidation. 2. Measurements of H2O2 and acid-soluble oxidation products during [1-14C]palmitoyl-CoA oxidation by purified peroxisomes revealed that the number of acetyl units released per molecule of palmitoyl-CoA oxidized rapidly decreased with increasing unbound palmitoyl-CoA concentrations. Structural damage to the peroxisomes caused by detergents or other treatments also decreased the number of acetyl units released. Under conditions where oxidation proceeded linearly with time the theoretical maximum of 5 acetyl units released per molecule of palmitoyl-CoA oxidized [Lazarow (1978) J. Biol. Chem. 253, 1522—1528] was never reached. 3. Expressed in terms of acetyl units produced and measured at low unbound-palmitoyl-CoA concentrations, mitochondrial oxidation was 10—20-fold higher than peroxisomal oxidation. 4. ATP stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. The ATP effect required the presence of Mg2+ and was lost when peroxisomal membranes were disrupted by Triton X-100 or high concentrations of unbound palmitoyl-CoA. 5. Disruption of peroxisomes by detergents, freeze—thawing, osmotic or mechanical treatment did not stimulate palmitoyl-CoA oxidation in the presence of ATP, indicating that peroxisomal fatty-acid-CoA oxidation was not latent. In the absence of ATP, Triton X-100 stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold.

The nuclear 3,5,3'-triiodothyronine receptor in human leukaemic cell lines

1984 ◽  
Vol 105 (3) ◽  
pp. 429-432 ◽  
Author(s):  
Juan Bernal ◽  
Leif C. Andersson
Keyword(s):  
Growth Hormone ◽  
Cell Line ◽  
Cell Lines ◽  
Rat Liver ◽  
Association Constant ◽  
Erythroid Differentiation ◽  
Leukaemic Cell ◽  
Cells In Culture ◽  
Gh Cells ◽  
High Concentrations

Abstract. The 3,5,3'-triiodothyronine (T3) receptor has been studied in a series of continuously growing human leukaemic cell lines. High concentrations of receptor were found in the erythroblastoid cell line K-562. T3 was bound to the nuclei of these cells with an association constant of 3.4 × 109 m−1, and capacity 104 fmol/100 μg DNA, or 8700 molecules/nucleus. This capacity is comparable to that of rat liver or growth hormone producing cells (GH cells) in culture, and suggests that the K-562 cell line could be a useful model for the study of T3 action on erythroid differentiation.

Hepatic uptake of intact glutathione S-conjugate, inhibition by organic anions, and sinusoidal catabolism

1993 ◽  
Vol 265 (3) ◽  
pp. G547-G554
Author(s):  
C. A. Hinchman ◽  
A. T. Truong ◽  
N. Ballatori
Keyword(s):  
Guinea Pig ◽  
Rat Liver ◽  
Marked Decrease ◽  
Hepatic Uptake ◽  
Half Time ◽  
High Concentrations ◽  
Rat Livers ◽  
Dose Dependent ◽  
Uptake And Metabolism ◽  
Guinea Pig Liver

To identify potential mechanisms for hepatic removal of circulating glutathione (GSH) conjugates, uptake and metabolism of S-2,4-dinitrophenylglutathione (DNP-SG) were examined in isolated perfused livers from rat and guinea pig. Guinea pig livers perfused with 5 mumol of DNP-SG in a recirculating system (50 microM initial concn) rapidly cleared the conjugate from the perfusate (half time 3.7 min), whereas clearance was considerably slower in rat liver (half time 35 min). Disappearance of DNP-SG from the perfusate was accompanied by a simultaneous appearance of DNP-SG and its metabolites in bile. Addition of acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), to the perfusate resulted in a marked decrease in DNP-SG clearance by guinea pig liver but had no effect in rat liver, suggesting that in the guinea pig this process is largely dependent on sinusoidal gamma-GT activity. However, even in the presence of acivicin, rat and guinea pig livers removed nearly one-half of the administered DNP-SG from the recirculating perfusate over 30 min. High concentrations of DNP-SG were found in bile (up to 3.7 mM), indicating that the liver is capable of transporting the intact conjugate from the circulation. When rat livers were perfused with higher concentrations of DNP-SG (100 and 250 microM), biliary excretion of DNP-SG increased dose dependently, with concentrations in bile reaching 10 mM at the higher dose. This was accompanied by a dose-dependent choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fractionation of rat liver mitochondrial components after short treatments with triton x-100

1982 ◽  
Vol 14 (10) ◽  
pp. 933-940 ◽  
Author(s):  
M.C. Barbero ◽  
E. Rial ◽  
J.J. Otamendi ◽  
J.I.G. Gurtubay ◽  
F.M. Goni
Keyword(s):  
Rat Liver ◽  
Triton X
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